Method of treating eye disease using glycosylated vegf decoy receptor fusion protein

ABSTRACT

The present application describes an isolated nucleic acid molecule encoding a polypeptide capable of synchronously binding VEGF polypeptide and placenta growth factor (PIGF) polypeptide comprising a nucleotide sequence encoding a VEGFR1 component.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present application relates to making a chimeric molecule that isused to prevent blood vessel formation as a form of treating cancer ortreating eye disease.

2. Description of the Background

Vascular endothelial growth factor-A (VEGF-A) is a critical regulator oftumor angiogenesis, mainly through the activation of its primaryreceptor, VEGF receptor 2 (VEGFR2). VEGF-A is expressed in most tumorcells and corresponding stromal cells throughout every stage of tumorprogression, while VEGFR2 is highly expressed in growing tumor vessels,leading to the formation of structurally and functionally malformedtumor blood vessels. VEGF-A specifically binds to the secondimmunoglobulin (Ig) homology domain (D2) of the extracellular region ofVEGFR2, resulting in activation of pro-angiogenic signalling. For thepast decade, much effort has been devoted to targeting thisVEGF-A/VEGFR2 signalling pathway using monoclonal antibodies, solubledecoy receptor fusion proteins, and small molecular inhibitors in cancerpatients. While the current therapeutic blockade of VEGF-A/VEGFR2signalling provides clinical benefits, the anti-cancer effect is modestand transient, eventually giving rise to acquired resistance through theactivation of alternative pro-angiogenic pathways and furtherrecruitment of pro-angiogenic cells such as tumor-associated macrophages(TAM). These limitations highlight current unmet needs inanti-angiogenic cancer treatment strategies, which must be addressed forsuccessful therapy development.

Many ocular diseases are also associated with abnormal angiogenesis andup-regulated VEGF. Particularly, exudative age-related maculardegeneration (AMD) is one of the most important causes of blindness indeveloped countries and the most clinically critical subtype of AMD. Inexudative AMD, the macular region rapidly deteriorates due to abnormalangiogenesis, termed “choroidal neovascularization (CNV)” which arisesfrom the choriocapillary across the retinal pigment epithelium (RPE) andBruch's membrane to subretinal space of macula. CNV is also a majorcomplication that threatens the vision of patients with various retinaldegenerative and inflammatory diseases, including pathologic myopia andocular histoplasmosis. Angiogenesis is normally a compensatory mechanismof our body in pathologic situations such as coronary collateralformation and wound healing process. And this mechanism is alsotriggered by an oxygen insufficiency state as known as “hypoxia”.Hypoxic state stimulates hypoxic inducible factors (HIFs) including VEGFand it plays a crucial role in angiogenesis. In addition, eyes with highconcentration of VEGF suffer from leakage from retinal vessels, andsubsequently, macular edema develops. Therefore, VEGF may be atherapeutic target for ocular diseases associated with abnormalangiogenesis and vascular leakage such as exudative AMD, diabeticretinopathy, retinopathy of prematurity, neovascular glaucoma, cornealneovascularization, retinal vein occlusion and macular edema due todiabetic retinopathy or retinal vein occlusion.

VEGF-A binds to both VEGFR1 and VEGFR2. The binding affinity of VEGFR1to VEGF-A (<10˜20 pM) is much higher than that of VEGFR2 (<100˜125 pM).In addition, VEGFR1 is a receptor for other pro-angiogenic ligands,VEGF-B and placental growth factor (PlGF), which have recently beenhighlighted as alternative targets for anti-angiogenic therapy. Becauseof its ability to bind multiple pro-angiogenic ligands, VEGFR1 has beenconsidered as a potential backbone for the development of a novel decoyreceptor fusion protein for therapeutic purposes. However, theefficiency of a decoy receptor fusion protein which consisted of thefirst 3 Ig domains of VEGFR1 fused with the Fc region of IgG1(VEGFR1-Fc) proved unsatisfactory, due to non-specific binding to theextracellular matrix (ECM) attributed to the abundant positively chargedresidues in the third Ig domain (VEGFR1 D3) and its high isoelectricpoint (pI) value. Nonetheless, this finding inspired the invention ofVEGF-Trap (Aflibercept from Regeneron), consisting of VEGFR1 D2 andVEGFR2 D3 fused to IgG1 Fc. By switching VEGFR1 D3 to VEGFR2 D3, the netpI of VEGF-Trap was decreased, resulting in less ECM binding and animproved pharmacokinetic (PK) profile compared to VEGFR1-Fc. However,because VEGFR2 D3 was used instead of VEGFR1 D3, the high-affinitybinding of VEGF-A and PlGF was disturbed. Hence, the important issue tobe addressed now is how to incorporate VEGFR1 D3 into a decoy receptorwhile minimizing non-specific ECM binding.

Glycosylation is a post-translational modification that results in theaddition of carbohydrate chains to specific asparagine (N-linkedglycosylation) or serine/threonine (O-linked glycosylation) residues.Glycosylation of secreted and membrane proteins affects theirbiochemical and biological properties. It usually provides a negativecharge and increases solubility, thus diminishing non-specific bindingto the ECM. Moreover, glycosylation grants resistance to proteolysis andextended serum half-life, enhancing a protein's PK profile.Glyco-engineered therapeutic proteins such as Aranesp (erythropoietin)from Amgen and Gazyva (obinutuzumab) from Genentech are good examplesthat exploited these advantages.

Here, we developed a novel VEGF decoy receptor fusion protein,VEGF-Grab. Parental VEGFR1-Fc (VEGFR1 D2-D3 fused to Fc) was used as abackbone, and new potential glycosylation sites were introduced into thepositively-charged patch of VEGFR1 D3 by site-directed mutagenesis. Thisengineered VEGF-Grab showed significantly improved decoy efficiency anda dramatic decrease in net pI, thus attenuating non-specific ECM bindingand enhancing PK profiles. Thus, VEGF-Grab strongly suppressed tumorangiogenesis, progression, and metastasis via effective capturing ofthree VEGFR1 ligands, VEGF-A, VEGF-B, and PlGF. Furthermore, we show theintravitreal therapeutic efficacy of VEGF-Grab3 to regress new vesselsin laser-induced CNV and oxygen-induced retinopathy (OIR) murine models,which are reliable methods in predicting the therapeutic value ofanti-VEGF therapies now approved for treating AMD and diabeticretinopathy.

SUMMARY OF THE INVENTION

Anti-angiogenic therapies targeting vascular endothelial growth factor A(VEGF-A) have been commonly used in clinics to treat cancers andage-related macular degeneration (AMD). However, its clinical efficacyhas been limited, with drawbacks including acquisition of resistance andactivation of compensatory pathways resulting from elevated circulatingVEGF-B and placenta growth factor (PlGF). To bypass these disadvantages,we developed a novel glycosylated soluble decoy receptor fusion protein,VEGF-Grab, which can neutralize VEGF-A, VEGF-B, and PlGF. VEGF-Grab hasthe second and third immunoglobulin (Ig)-like domains of VEGF receptor 1(VEGFR1) fused to IgG1 Fc, with three potential glycosylation sitesintroduced into the third Ig-like domain of VEGF-Grab by mutagenesis.Compared to VEGF-Trap, VEGF-Grab showed more potent decoy activityagainst VEGF and PlGF, mainly attributed to the VEGFR1 backbone. Mostimportantly, the negatively charged O-glycans attached to the thirdIg-like domain of VEGFR1 counterbalanced the originally positivelycharged VEGFR1 backbone, minimizing non-specific binding of VEGF-Grab tothe extracellular matrix, a n d resulting in greatly improvedpharmacokinetic profile. These advancements led to stronger and moredurable anti-angiogenic and anti-tumor efficacy as compared toVEGF-Trap, while toxicity profiles were comparable to VEGF-Trap.Collectively, our results highlight VEGF-Grab as a promising therapeuticcandidate for further clinical drug development.

In one aspect, the present invention is drawn to an isolated nucleicacid molecule encoding a polypeptide capable of synchronously bindingVEGF polypeptide and placenta growth factor (PIGF) polypeptidecomprising a nucleotide sequence encoding a VEGFR1 component. The VEGFR1component may include the second and third immunoglobulin (Ig)-likedomains. The at least one encoded positive amino acid residue in atleast one domain of VEGFR1 may be mutated to a negatively chargedresidue. The domain may be the third domain. And the amino acid residuemay be on the β1-β2 loop, which may include nucleic acid positions 397to 432 of SEQ ID NO:1, which corresponds to amino acid residues 133 to144 of SEQ ID NO:2, or β3-β4 loop, which comprises nucleic acidpositions 490 to 522 of SEQ ID NO:1, which corresponds to amino acidresidues 164 to 174 of SEQ ID NO:2. Also, the at least one encodedpositive amino acid residue in at least one domain of VEGFR1 may bemutated so as to produce a glycosylation site. The at least one encodedpositive amino acid residue in at least one domain of VEGFR1 may bemutated so as to produce a decrease in net pI of the encodedpolypeptide. The residue to be mutated may be R135 residue on the β1-β2loop, K138 residue on the β1-β2 loop, or R172 residue on the β3-β4 loopon the third domain. The VEGF may be VEGF-A or VEGF-B. Optionally, theVEGFR1 component may be operatively linked to a nucleotide sequenceencoding a multimerizing component.

In another aspect, the invention is directed to an isolated nucleic acidmolecule comprising a nucleotide sequence encoding: (a) VEGF-Grab1; (b)VEGF-Grab2; or (c) VEGF-Grab3. In yet another aspect, the invention isdrawn to a nucleic acid vector, which includes any of the nucleic acidmolecule described above. The vector may be viral vector. Or, the vectormay be an expression vector that includes any of above-described nucleicacid molecule, wherein the nucleic acid molecule may be operativelylinked to an expression control sequence.

In yet another aspect, the invention is directed to a method ofgenerating a polypeptide capable of synchronously binding VEGFpolypeptide and placenta growth factor (PIGF) polypeptide comprising aVEGFR1 component in a patient, comprising administering to the patientthe nucleic acid vector described above.

In yet another aspect, the invention is drawn to a host-vector systemfor the production of a polypeptide, which includes the expressionvector described above, in a suitable host cell. The suitable host cellmay be a bacterial cell, yeast cell, insect cell, or mammalian cell.

In another aspect, the invention is drawn to a method of generating apolypeptide capable of synchronously binding VEGF polypeptide andplacenta growth factor (PIGF) polypeptide comprising a VEGFR1 componentin a patient, comprising administering to the patient the cell describedabove.

In another aspect, the invention is drawn to a method of producing apolypeptide which steps include growing cells of the host-vector systemdescribed, under conditions permitting production of the polypeptide andrecovering the polypeptide so produced.

In yet another aspect, the invention is drawn to a polypeptide encodedby any of the isolated nucleic acid molecule described above. Thepolypeptide may be wherein the VEGFR1 component comprises the second andthird immunoglobulin (Ig)-like domains. The polypeptide may be whereinat least one positive amino acid residue in at least one domain ofVEGFR1 may be mutated to a negatively charged residue. The domain may bethe third domain. The amino acid residue may be on the β1-β2 loop, whichmay include amino acid residues 133 to 144 of SEQ ID NO:2, or β3-β4loop, which may include amino acid residues 164 to 174 of SEQ ID NO:2.The at least one positive amino acid residue in at least one domain ofVEGFR1 may be mutated so as to include a glycosylation site. Thepolypeptide may be glycosylated. Further, the polypeptide may besialylated. In the polypeptide discussed above, at least one positiveamino acid residue in at least one domain of VEGFR1 may be mutated so asto produce a decrease in net pI of the polypeptide. The residue to bemutated may be R135 residue on the β1-β2 loop, K138 residue on the β1-β2loop, or R172 residue on the β3-β4 loop on the third domain.

In another aspect, the invention is drawn to a method of blocking bloodvessel growth in a mammal comprising administering to the mammal in needthereof an effective amount of the polypeptide described above. Themammal may be human.

The blood vessel growth may occur in the eye to cause a medicalcondition that affects sight. The medical condition may be age-relatedmacular degeneration, exudative age-related macular degeneration,choroidal neovascularization, pathologic myopia, diabetic retinopathy,diabetic macular edema, retinal vein occlusion, retinopathy ofprematurity or neovascular glaucoma. The choridal neovascularization maybe myopic choroidal neovascularization, traumatic choroidalneovascularization, uveitic choroidal neovascularization, ocularhistoplasmosis, or idiopathic choroidal neovascularization.

In yet another aspect, the invention is drawn to a method of inhibitingVEGF and/or PIGF activities in a mammal comprising administering to themammal an effective amount of any of the polypeptides described above.The mammal may be human.

In another aspect, the invention is drawn to a method of attenuating orpreventing tumor growth in a mammal, comprising administering to asubject in need thereof a therapeutically effective amount of thepolypeptides described above. The mammal may be human.

In yet another aspect, the invention is drawn to a method of suppressingmetastasis in a mammal, comprising administering to a subject in needthereof a therapeutically effective amount of the polypeptides describedabove. The mammal may be human.

In another aspect, the invention is drawn to a method of attenuating orpreventing tumor growth in a mammal, comprising administering to asubject in need thereof a therapeutically effective amount of any of thepolypeptides described above and a cytotoxic therapeutic agent. Themammal may be human. And, the cytotoxic therapeutic agent may be withoutlimitation cisplatin.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given herein below, and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein;

FIGS. 1A-1G show generation and characterization of VEGF-Grab. FIG. 1A,Schematic diagram of VEGF-Grabs (VEGF-Grab1, VEGF-Grab2 and VEGF-Grab3;hereinafter referred to as G1, G2, and G3) and VEGF-Trap (VT). Mutatedresidues in VEGF-Grabs are indicated by brown at the lower panel. FIG.1B, Electrostatic potential of D2-D3 domain in VEGFR1 (left) and VEGFR2(right). Positively and negatively charged residues are colored in blueand red, respectively. FIG. 1C, Model structure of VEGF-A/VEGFR1 D2-D3complex. VEGF-A dimer is colored in yellow and green. VEGFR1 D2 and D3are colored in tan and mustard. Residues indicated by blue stick aretarget sites for mutagenesis. FIG. 1D, SDS-PAGE analysis of G1 and G3 inreduced (R) and non-reduced (NR) conditions. E-G, Binding affinities ofG1, G3, and VT for VEGF-A (FIG. 1E), PlGF (FIG. 1F), or VEGF-B (FIG. 1G)*p<0.05 G1 vs VT; #p<0.05 G3 vs VT. For each group, n=3. Values aremean±SD.

FIG. 2 shows secondary structure of original VEGFR1-Ig3. β strands areshown as arrows and cylinders (green). Residues on β1-β2 and β3-β4 loopsof VEGFR1-D3 are displayed as bold.

FIGS. 3A-3K show that VEGF-Grab3 exhibits low ECM binding and prolongedpharmacokinetic profile. FIG. 3A, Isoelectric point analysis of G1, G3,and VT. Red lines on each band were used to analyse net pI. FIG. 3B.Comparison of pI for each protein. Net pI of each protein was the meanpI of each isoform denoted as lines. FIG. 3C, PNGase F digestion toremove N-linked glycan. FIG. 3D, Analysis of O-linked glycosylation atSerine135 of G3. FIG. 3E, Schematic diagram for glycosylated sitesanalysed by mass spectrometry. Occupied N-glycosylation sites (red);occupied O-glycosylation sites (blue); unoccupied N- or O-glycosylationsites (grey). F and G, PK profiles analysis of VT (FIG. 3F) and G3 (FIG.3G) at varying doses. FIG. 3H, Comparison of area under the curve (AUC)of VT and G3. I and J, Tissue distribution of G3 and VT 48 hr aftersubcutaneous injection (4 mg/kg). FIG. 3I, Accumulated VT and G3 intumor. FIG. 3J, Relative accumulated levels of VT and G3 in liver andkidney compared to tumor. FIG. 3K. Analysis of in vitro ECM bindingaffinities with Matrigel-coated plates. *p<0.05 G1 vs VT; #p<0.05 G3 vsVT. Each group, n=3. Values are mean±SD.

FIGS. 4A-4H show that VEGF-grabs inhibit EC survival, migration, andtube formation via suppression of VEGF signalling pathway. FIGS. 4A-4C,Inhibition of VEGF-A-induced phosphorylation of VEGFR2 and ERK in HUVECsby the treatment of G1, G3, and VT. Immunoblotting (FIG. 4A) andquantification (FIG. 4B and FIG. 4C). FIG. 4D, Cell survival assay withHUVECs after G1, G3, and VT treatment (0.35, 0.7, 3.5, 7, 35, 70 nM) inthe presence of VEGF-A (0.2 nM). FIG. 4E and FIG. 4F, Cell migrationassay with HUVECs in the presence of VEGF-A and indicated proteins.Images (FIG. 4E) and quantification (FIG. 4F) of migration area. Woundhealing areas are indicated in red. FIG. 4G and FIG. 4H, Images (FIG.4G) and quantification (FIG. 4H) for tube formation assay with HUVECs inthe presence of VEGF-A and indicated proteins. *p<0.05 vs control;#p<0.05 G3 vs VT. Each group, n=3. Values are mean±SD.

FIGS. 5A-5U show that VEGF-Grab3 effectively suppresses tumor growth,angiogenesis, and metastasis in LLC tumors. FIGS. 5A-5N, Mice weretreated with proteins on the indicated days (arrows). FIG. 5A and FIG.5C, Comparison of tumor growth (FIG. 5A) and tumor weights (FIG. 5C).FIG. 5B and FIG. 5D, Images (FIG. 5B) and quantification (FIG. 5D) ofintratumoral necrotic area stained with H&E. Dotted line demarcatesintratumoral necrosis. FIG. 5E and FIG. 5F, Images (FIG. 5E) andquantification (FIG. 5F) of CD31⁺ blood vessels in the peri- andintratumoral area. FIG. 5G, Images showing cytokeratin⁺ tumor cellmetastasis (red) in inguinal LNs. Each indicated region (squares) ismagnified in the lower panel. FIG. 5H and FIG. 5I, Quantifications oflymphatic vascular densities (LVD) (FIG. 5H) and cytokeratin⁺ tumor cellmetastasis (FIG. 5I). FIG. 5J and FIG. 5K, Images (FIG. 5J) andquantifications (FIG. 5K) of Hypoxyprobe⁺ hypoxic areas (green) intumors. FIG. 5L and FIG. 5M, Images (FIG. 5L) and quantifications (FIG.5M) of CD11b⁺ myeloid cells (red) in tumor. FIG. 5N, Comparisons of mRNAexpression levels of various genes in intratumoral tissue aftertreatment with G3 and VT. Values indicate fold changes over controltumors. FIGS. 5O-5R, Comparative dose responses of VT and G3 on tumorgrowth and metastasis. LLC tumor-bearing mice were treated with eitherVT, or G3 at the indicated days (arrows), respectively. FIG. 5O and FIG.5P, Comparison of tumor growth after VT (FIG. 5O) or G3 (FIG. 5P)treatment. FIG. 5Q and FIG. 5R, Images (FIG. 5Q) and quantification(FIG. 5R) of cytokeratin⁺ tumor cell metastasis in inguinal LNs. Scalebars, 100 μm. *p<0.05 vs control; #p<0.05 G3 vs VT. FIGS. 5S-5U,Combination therapy of cisplatin (green arrows, at day 9 after tumorimplantation) with either VT, or G3 at the indicated days (blackarrows). FIG. 5S, Comparison of LLC tumor growth. Images (FIG. 5T) andcomparison (FIG. 5U) of Caspase3⁺ apoptotic cells (red) in intratumoralarea. For each group n=5. Values are mean±SD. Scale bars, 100 μm.*p<0.05 vs cisplatin; #p<0.05 cis+G3 vs cis+VT.

FIGS. 6A-6I show that VEGF-Grab3 delays tumor growth and suppressesneovessel formation and metastasis in a spontaneous breast cancer model.FIGS. 6A-6I, Female MMTV-PyMT mice (12-weeks old) receivedintraperitoneal-injections of VT, or G3 (25 mg/kg) twice per week for 3weeks. FIG. 6A, Tumor sections stained with H&E. Invasive tumor cells(Inv), early carcinoma lesions (Ea), and surrounding adipose tissue(Adi) are denoted by dotted lines. FIG. 6B, Comparison of volumes oftumor nodules. Lines denote mean values. FIG. 6C and FIG. 6D, Images(FIG. 6C) and comparison (FIG. 6D) of CD31⁺ blood vessels in the peri-and intratumoral areas. FIG. 6E and FIG. 6F, Images (FIG. 6E) andcomparison (FIG. 6F) of Caspase3⁺ apoptotic cells (red) in tumor. FIG.6G, Images showing cytokeratin⁺ tumor cell metastasis (red) in axillaryLNs. FIG. 6H and FIG. 6I, Quantifications of LVD (FIG. 6H) andcytokeratin⁺ tumor cell metastasis (FIG. 6I) in axillary LNs. Unlessotherwise noted, for each group, n=4. Values are mean±SD. Scale bars,100 μm. *p<0.05 vs control. #p<0.05 G3 vs VT.

FIGS. 7A-D show that VEGF-Grab3 exerts more durable suppression of tumorangiogenesis. FIGS. 7A-7D, tumor vessel regrowth after VT or G3treatment. FIG. 7A. Experimental Scheme. Mice were treated with proteinson the indicated days (green arrows). Treatment was withdrawn, andanalysed at D17 and D19 (blue arrows). FIGS. 7B-7D, Changes in thevascularity after treatment with either VT, or G3. White arrowheadsindicate representative new vascular sprouts. Images (FIG. 7B) andquantifications of CD31⁺ blood vessels (FIG. 7C) and sprouts numbers(FIG. 7D). *p<0.05 VT D17 vs VT D19.

FIGS. 8A-8B show that a single intravitreal injection of 50 μgVEGF-Grab3 suppresses choroidal neovascularization at sites of ruptureof Bruch's membrane. FIG. 8A. Representative confocal microscope imagesof the anti-PECAM-1/CD31 antibody-stained CNV lesions in the choroidflat mounts from laser-induced CNV in mice treated with PBS (Left),VEGF-Grab3 (middle) and Aflibercept (Right). CNVs were significantlyabolished by the VEGF-Grab3 or Aflibercept treatment compared toPBS-treatment. Scale bar=100 μm. FIG. 8B. Bar graph showing the size ofCNVs area developed in the laser-induced control PBS injected eyes,Aflibercept-treated eyes and VEGF-Grab3-treated eyes. Data are expressedas means±standard error of the mean (SEM). Measurement of the area ofCNV by image analysis confirmed that there was significantly lessneovascularization in eyes treated with VEGF-Grab3 (60 rupture sites) orAflibercept (60 rupture sites) compared to those treated with PBS (60rupture sites). Statistical differences between means were determined byanalysis of variance with one-way followed by the Bonferroni test (*P<0.01).

FIGS. 9A-9B show that intravitreal injection of VEGF-Grab3 reduced thevascular density of OIR model mice. FIG. 9A. Representative magnifiedconfocal microscope images of OIR model mice. The density of vasculaturewas significantly reduced by the VEGF-Grab3 treatment compared toPBS-treatment. FIG. 9B. Bar graph showing VEGF-Grab3 increases the areaof avascular retina at P17 in OIR model mice. Data are expressed asmeans±standard error of the mean (SEM). The vascular intensity ofVEGF-Grab3 treated mice significantly decreased compared with thePBS-injected control eyes. (P<0.01).

FIG. 10 shows sequence alignment of VEGF-Grab1, VEGF-Grab3, andVEGF-Trap. Amino acid sequences of G1, G3, and VT were aligned usingCLUSTALW. Signal sequences, D2, D3, and Fc domains are shown above thesequence alignment. N-linked glycosylation sites detected by massspectrometry in all proteins are highlighted by the red box. The mutatedsite occupied by O-linked glycosylation is highlighted by the blue box.The mutated sites for glycosylation that are unoccupied by glycans arehighlighted by the grey box. The peptide fragments analysed by MSspectrometry after trypsin digestion are shown below the sequence asbrown lines (See FIG. 11A for additional description).

FIGS. 11A-11B show analysis of N-linked glycosylation for VEGF-Grabs andVEGF-Trap. FIG. 11A, List of peptides possibly identified fromPNGaseF/trypsin digestion of VEGF-Grabs and VT that encompass apotential site of N-glycosylation. If an Asn residue is N-glycosylatedat a particular position, that residue will be converted to Asp afterPNGase digestion, resulting in a mass gain of 0.984 Da. In contrast, Asnresidues unoccupied by N-glycans are unaffected by deglycosylation.Thus, by tracking the mass of deglycosylated tryptic peptide with LC/MS,the occupied or unoccupied states of N-glycosylation sites can bedetermined. Detected peptides are indicated as ✓. Undetected peptidesare indicated as x. N/A indicates not available. FIG. 11B, Relativeabunda nces of each N-glycan class in VT, G1, and G3:fucosylated/sialylated complex/hybrid (C/H-Fuc&Sia); sialylatedcomplex/hybrid (C/H-Sia); fucosylated complex/hybrid (C/H-Fuc);undecorated complex/hybrid (C/H); high mannose (HM).

FIG. 12 shows analysis of O-linked glycosylation in VEGF-Grab1. TandemMS spectrum of G1 glycopeptide. O-linked glycosylation at the Serinel35was detected. Extensive peptide and glycan fragmentation enabledcomplete site- and structure-specific assignment.Square=N-acetylhexosamine, circle=hexose, and diamond=sialic acid.

FIG. 13 shows that VEGF-Grab3 displays low binding to tumor ECM. Controltumor sections which were not treated with any protein therapeutics,were stained with 100 nM of VR1-Fc, VT, or G3 and subsequentlyvisualized using the anti-human Fc-cy3 antibody to detect non-specificbinding (red) of these molecules to tumor ECM. Collagen type IV (green)is a collagen primarily found in the basal lamina of the ECM. Scalebars, 100 μm.

FIG. 14 shows pharmacokinetic profile of VEGF-Grab1 and VEGF-Grab3.Serum levels of G1 after subcutaneous injection of 4 mg/kg. Blood wassampled at 0, 1, 2, 4, 8, 12, 24, 48, and 96 hr, and analysed by ELISA.

FIGS. 15A-15B show tissue accumulation of VEGF-Trap and VEGF-Grab3.FIGS. 15A and 15B, VT and G3 levels in tissues after subcutaneousinjections of VT (4 mg/kg) and G3 (4 mg/kg) into LLC tumor-bearingC57BL/6J mice. Tissues were harvested after 48 hr, lysed in lysis buffer(Lysis buffer 6, R&D), quantified by Bradford assay, and analysed byELISA for liver (FIG. 15A) and kidney (FIG. 15B). *p<0.05 G3 vs VT.Values are mean±SD.

FIGS. 16A-16G show that VEGF-Grabs or VEGF-Trap have no effect on VEGFR2signalling, EC survival, migration, and tube formation in the absence ofVEGFA. FIGS. 16A-16C, VEGFR2 and ERK phosphorylation in HUVECs aftertreatment with G1, G3, and VT (2 μg/ml, 14 nM, respectively) in thepresence or absence of VEGFA (50 ng/ml, 1 nM). Immunoblotting (FIG. 16A)and quantification (FIG. 16B and FIG. 16C). und. indicates undetected.FIG. 16D and FIG. 16E, Cell migration assay with HUVECs in the presenceor absence of VEGFA (50 ng/ml, 1 nM) and indicated proteins (2 μg/ml, 14nM). The amount of wound healing was monitored after 12 hr. Woundhealing areas are indicated in red. Images (FIG. 16D) and quantification(FIG. 16E) of migration area. FIG. 16F and FIG. 16G, Tube formationassay with HUVECs in the presence or absence of VEGFA (50 ng/ml, 1 nM)and indicated proteins (2 μg/ml, 14 nM). Images (FIG. 16F) andquantification (FIG. 16G) of tube formation. Scale bars, 100 μm.

FIGS. 17A-17B show dose-dependent inhibition of VEGFR2 signalling withanti-VEGF therapy. FIGS. 17A-17B, HUVECs were cultured, starvedovernight, pre-treated with each protein (0.125 μg/ml (0.875 nM), 0.25μg/ml (1.75 nM), 0.5 μg/ml (3.5 nM), and 1 μg/ml (7 nM)) for 15 min, andtreated with VEGFA (50 ng/ml, 1 nM) for 10 min. Cells were lysed andindicated proteins were immunoblotted to detect the activation of VEGFR2and ERK1/2 signalling. FIG. 17A, Immunoblotting showing dose-dependentinhibition of VEGFR2 and ERK1/2 phosphorylation after treatment withVEGF-Grabs or VT. FIG. 17B, Comparisons of VEGFR2 and ERK1/2phosphorylation after treatment with VEGF-Grabs or VT.

FIG. 18 shows histologic analyses of vital organs after anti-VEGFtherapy. After a 2-week treatment with either control, VT, or VEGF-Grabs(25 mg/kg) in LLC tumor-bearing mice, indicated organs were sampled andsectioned for histologic analysis. Images show tissue sections ofindicated organs stained with H&E. Scale bars, 100 μm.

FIG. 19 shows that VEGF-Grab effectively suppresses the growth ofestablished bulky macroscopic tumors. After the volume of tumor exceeded500 mm³, either control, VT, or G3 were treated to LLC tumor bearingmice on the indicated days (arrows). Comparison of LLC tumor growth.

FIGS. 20A-20B show histological analyses of the kidney and liver afterdose dependent anti-VEGF therapy. After treatment with either control,VT (5, 10, 25, and 50 mg/kg), or G3 (5, 10, 25, and 50 mg/kg) in LLCtumor-bearing mice, kidney and liver were sampled and sectioned forhistologic analysis. Images show tissue sections of kidney (FIG. 20A)and liver (FIG. 20B) stained with H&E. Scale bars, 100 μm.

FIG. 21 shows that combination therapy of VEGF-Grab3 and Cisplatinenhances intratumoral apoptosis. LLC tumor-bearing mice receivedinjections of cisplatin (10 mg/kg) 9 days after tumor implantation incombination with either control, VT, or G3 (25 mg/kg) every 2 days.After combination therapy, Caspase 3+ cells (Red) mostly overlap withPan-cytokeratin⁺ cells (Green) which are epithelial cell derived LLCtumor, but not with CD31⁺ cells (Blue), suggesting apoptotic cells aremore likely tumor cell.

DETAILED DESCRIPTION OF THE INVENTION

In the present application, “a” and “an” are used to refer to bothsingle and a plurality of objects.

As used herein, “about” or “substantially” generally provides a leewayfrom being limited to an exact number. For example, as used in thecontext of the length of a polypeptide sequence, “about” or“substantially” indicates that the polypeptide is not to be limited tothe recited number of amino acids. A few amino acids add to orsubtracted from the N-terminus or C-terminus may be included so long asthe functional activity such as its binding activity is present.

As used herein, administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

As used herein, “amino acid” and “amino acids” refer to all naturallyoccurring L-┌-amino acids. This definition is meant to includenorleucine, ornithine, and homocysteine.

As used herein, in general, the term “amino acid sequence variant”refers to molecules with some differences in their amino acid sequencesas compared to a reference (e.g. native sequence) polypeptide. The aminoacid alterations may be substitutions, insertions, deletions or anydesired combinations of such changes in a native amino acid sequence.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Also included within the scope of the invention areproteins or fragments or derivatives thereof which exhibit the same orsimilar biological activity and derivatives which are differentiallymodified during or after translation, e.g., by glycosylation,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, and so on.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative amino acid sequence. Immediately adjacent to an amino acid meansconnected to either the ┌-carboxy or ┌-amino functional group of theamino acid.

Deletional variants are those with one or more amino acids in the nativeamino acid sequence removed. Ordinarily, deletional variants will haveone or two amino acids deleted in a particular region of the molecule.

As used herein, “antagonist” refers to a ligand that tends to nullifythe action of another ligand, as a ligand that binds to a cell receptorwithout eliciting a biological response.

It is also contemplated that fusion proteins be labeled with adetectable label, such as radioisotope, fluorescent tag, enzymatic tag,or a chemiluminescent tag to determine ligand-receptor bindinginteraction. As such, assay systems employing the chimeric molecule isalso contemplated.

As used herein, “carriers” include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe pharmaceutically acceptable carrier is an aqueous pH bufferedsolution. Examples of pharmaceutically acceptable carriers includewithout limitation buffers such as phosphate, citrate, and other organicacids; antioxidants including ascorbic acid; low molecular weight (lessthan about 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

As used herein, “consisting essentially of” when used in the context ofa nucleic acid sequence refers to the sequence that is essential tocarry out the intended function of the amino acid encoded by the nucleicacid.

As used herein, “effective amount” is an amount sufficient to effectbeneficial or desired clinical or biochemical results. An effectiveamount can be administered one or more times. For purposes of thisinvention, an effective amount of an inhibitor compound is an amountthat is sufficient to palliate, ameliorate, stabilize, reverse, slow ordelay the progression of the disease state.

As used herein, “fragments” or “functional derivatives” refers tobiologically active amino acid sequence variants and fragments of thenative ligands or receptors of the present invention, as well ascovalent modifications, including derivatives obtained by reaction withorganic derivatizing agents, post-translational modifications,derivatives with nonproteinaceous polymers, and immunoadhesins.

As used herein, “host cell” includes an individual cell or cell culturewhich can be or has been a recipient of a vector of this invention. Hostcells include progeny of a single host cell, and the progeny may notnecessarily be completely identical (in morphology or in total DNAcomplement) to the original parent cell due to natural, accidental, ordeliberate mutation and/or change.

As used herein, “ligand” refers to any molecule or agent, or compoundthat specifically binds covalently or transiently to a molecule such asa polypeptide. When used in certain context, ligand may includeantibody. In other context, “ligand” may refer to a molecule sought tobe bound by another molecule with high affinity.

As used herein, “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, and so on. Preferably, the mammal is human.

As used herein “pharmaceutically acceptable carrier and/or diluent”includes any and all solvents, dispersion media, coatings antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, use thereofin the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form. A unit dosageform can, for example, contain the principal active compound in amountsranging from 0.5 μg to about 2000 mg. Expressed in proportions, theactive compound is generally present in from about 0.5 μg/ml of carrier.In the case of compositions containing supplementary active ingredients,the dosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

As used herein, “sample” or “biological sample” is referred to in itsbroadest sense, and includes any biological sample obtained from anindividual, body fluid, cell line, tissue culture, or other source,which may contain any PIGF or VEGF-A binding peptides, depending on thetype of assay that is to be performed. As indicated, biological samplesinclude body fluids, such as semen, lymph, sera, plasma, urine, synovialfluid, spinal fluid and so on. Methods for obtaining tissue biopsies andbody fluids from mammals are well known in the art.

As used herein, “subject” is a vertebrate, preferably a mammal, morepreferably a human.

As used herein, “synchronous” or “synchronously” binding refers to thebinding of the protein to two or more designated proteins simultaneouslyif the proteins are available for binding.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. “Palliating” a disease means that theextent and/or undesirable clinical manifestations of a disease state arelessened and/or the time course of the progression is slowed orlengthened, as compared to a situation without treatment.

As used herein, “vector”, “polynucleotide vector”, “construct” and“polynucleotide construct” are used interchangeably herein. Apolynucleotide vector of this invention may be in any of several forms,including, but not limited to, RNA, DNA, RNA encapsulated in aretroviral coat, DNA encapsulated in an adenovirus coat, DNA packaged inanother viral or viral-like form (such as herpes simplex, andadeno-structures, such as polyamides.

VEGF-Grab

In this study, we developed a novel glycosylated soluble decoy receptorfusion protein containing VEGFR1 D2-D3 and Fc, called VEGF-Grab, whichdemonstrates a prolonged PK profile and sequesters both VEGF and PlGF.Although VEGFR1 binds to VEGF-A and PlGF with higher affinity than doesVEGFR2, the development of therapeutic decoy proteins with the VEGFR1backbone has proven to be difficult thus far. The major reason behindthis was the high pI value VEGFR1 due to the positively charged residuesat the VEGFR1 D3 region, in particular, β1-β2 loop and/or β3-β4 loop.This causes non-specific ECM binding and poor PK profiles, leading to ashortened half-life, a subsequent decrease in efficacy, and even toxicside effects. In order to overcome this intrinsic property in VEGFR1 D3,we mutated three positive residues within VEGFR1 D3 loop that werepredicted to be irrelevant to ligand binding. These positive residueswere altered to become potential glycosylation sites (Ser, Thr or Asn).

Creating new decoy receptor fusion proteins using this glycosylationstrategy results in several advantages. First, ECM binding of VEGF-Grabis dramatically decreased by introducing these glycosylation sites intothe VEGFR1 D3 region. These sites counterbalance the positively chargedresidues with negatively charged residues or newly attached negativelycharged glycans. In addition, as shown by glycan analysis with massspectrometry, G3 contains increased sialylation compared with G1 or VT.Terminal sialic acid is critical for in vivo half-life of proteins sincethe asialo-glycoprotein receptors in the liver bind to nonsialylatedglycoproteins and remove them from the serum by endocytosis. This lowerECM binding and increased sialylation of G3 appears to facilitate anenhanced PK profile. Specifically, compared to VT, the AUC of G3 wereincreased by 1.7˜1.9-fold, suggesting that bioavailability of G3 aresuperior to VT. Second, G3 containing the VEGFR1 D2-D3 showed morepotent decoy activity against VEGF-A and PlGF, compared to VT; G3 bound1.5-fold and 6.7-fold higher to VEGF-A and PlGF as compared to VT. Thiswas evidenced by our in vitro experiments demonstrating strongsuppression of EC proliferation, migration, and tube formation aftertreatment with G3. Consistent results were observed in vivo, where G3showed much stronger anti-angiogenic, anti-tumor, and anti-metastaticeffects in both implanted and spontaneous tumor models compared to VT.The binding affinity of G3 to PlGF, which is critical for TAMrecruitment, was comparable to that of anti-PlGF antibody, resulting inthe decreased macrophage infiltration in G3-treated tumor compared tothose treated with VT. Considering that the LLC tumor model is known tobe relatively resistant to anti-VEGF therapy, these findings suggest thepossibility of overcoming resistance to anti-VEGF therapy by concurrentblockade of PlGF with VEGF-Grab. Third, G3 demonstrated improvements intoxicity profile as compared to the parental VEGFR1-Fc. Ascitesformation and mortality were reported with the use of VEGFR1-Fc due toits non-specific interaction with the ECM. During our animalexperiments, G3 was treated for long periods lasting 2-3 weeks, with nosigns of ascites formation or mortality (data not shown). Furthermore,histologic analyses of vital organs did not reveal any significantdifferences in comparison to VT (FIG. 18), implying that the toxicity ofparental VEGFR1-Fc can be overcome by introducing additionalglycosylation. However, adverse effects of systemic anti-VEGF therapyhave been reported, including delayed wound healing and hemorrhage.Recently, Sticky-trap, which locally inhibits angiogenesis, has beendeveloped and confirmed to have no systemic side effects. While thepotential side effects of G3 in clinics should be carefully studied atvarying dosages, time points, and after long-term treatment, it could befurther improved by adopting the Sticky-trap concept.

Currently, anti-VEGF agents are approved for clinical use in combinationwith chemotherapy for the treatment of various tumors. Here, we alsodemonstrated the potential application of VEGF-Grab as a candidate forcombinational chemotherapy with its additive and synergistic effects. Inour study, a single administration of VEGF-Grab3 demonstrated anequivalent outcome in vivo murine CNV and OIR model to aflibercept. AsVEGF-Trap (aflibercept) was also FDA-approved for the treatment ofage-related macular degeneration (AMD) exhibiting higher efficiency thana single approach inhibiting VEGF-A, indicating VEGF-Grab can be alsoapplicable to angiogenic ocular diseases including AMD. In clinicalpractice, monthly or bimonthly repetitive injections of anti-VEGF agentsneed to maintain a visual acuity in AMD patients. However, our enhancedPK profile suggests a probability of similar outcomes as afliberceptwith a less frequent dose regimen, which is consistent with therationale that a higher binding affinity could lead to increaseddurability.

In conclusion, our evidence suggests VEGF-Grab3 is a potent andeffective recombinant decoy for both VEGF and PlGF. Through the enhancedPK profile, VEGF-Grab3 showed durable suppression of tumor angiogenesis,growth, and metastasis. Clinical applicability of this novel fusionprotein should be explored through further preclinical and clinicalstudies.

Treatment of Ocular Diseases

Many ocular diseases are also associated with abnormal angiogenesis,vascular leakage and up-regulated VEGF. In one aspect, the inventiveglycosylated VEGF decoy receptor fusion protein may be administered to apatient with an eye condition associated with abnormal angiogenesis orvascular leakage. The patient may be a person suffering from unwantedneovascularization in the eye or macular edema.

Exudative Age-Related Macular Degeneration (AMD)

Exudative age-related macular degeneration (AMD) is one of the mostimportant causes of blindness in developed countries and the mostclinically critical subtype of AMD. In exudative AMD, the macular regionrapidly deteriorates due to abnormal angiogenesis, termed “choroidalneovascularization (CNV)” which arises from the choriocapillaris acrossthe retinal pigment epithelium (RPE) and Bruch's membrane to subretinalspace of macula. CNV is also a major complication that threatens thevision of patients with various retinal degenerative and inflammatorydiseases, including pathologic myopia. Additional CNV conditions mayinclude without limitation, myopic choroidal neovascularization,traumatic choroidal neovascularization, uveitic choroidalneovascularization such as ocular histoplasmosis, and idiopathicchoroidal neovascularization.

Myopic Choroidal Neovascularization

Myopic CNV is a disease of the retina where new, abnormal blood vesselsgrow into the retina in persons who are severely myopic (typically morethan minus six diopters). The disease is characterized by an abnormallyelongated eye with a physical stretching of the sclera, choroid, andretina resulting in degenerative and progressive changes. Thesedegenerative changes can induce rupture in the Bruch's membrane and thedevelopment of choroidal neovascularization. Similar mechanism appliesto traumatic CNV.

Uveitic Choroidal Neovascularization

Uveitis is an inflammation of the uvea. CNV is an uncommon complicationof uveitis associated with visual impairment that occurs more commonlyin forms affecting the outer retina-retinal pigment epithelium-choroidinterface, during periods of inflammatory activity, in association withpreretinal neovascularization, and in second eyes of patients withunilateral CNV.

Ocular Histoplasmosis

Ocular histoplasmosis and multifocal choroiditis and panuveitis (MCP)syndrome are examples of uveitis that are sometimes complicated by CNV.CNV is the main reason for vision deterioration in those ocularinflammatory diseases.

A fungus is inhaled early in life and causes a usually asymptomatic andself-limited infection throughout the body, including the lungs andchoroid (the vascular layer lining the retina). For unknown reasons,several decades after the initial infection, choroidal scars may developabnormal blood vessels (choroidal neovascularization) which leak fluidand blood. Distorted central vision and loss of reading vision occurswhen the leakage involves the macula. A goal of treatment is to preventchoroidal neovasculatization (CNV) from spreading into the macularcenter, or limit the size of and leakage from the CNV once it reachesthe macular center.

Idiopathic Choroidal Neovascularization

When new blood vessels originating from the choroid appear to arisespontaneously, without a known cause, the condition is referred to asidiopathic choroidal neovascularization. The new blood vessels mayproliferate beneath the retina's pigment epithelial layer, causing type1 neovascularization. They may penetrate the pigment epithelial layerand occupy the sub-retinal space beneath the sensory retina, causingtype 2 neovascularization. Regardless of underlying causes and locationsof growth, neovascularization results in vision loss.

Diabetic Retinopathy and Diabetic Macular Edema

Diabetic retinopathy develops in patients with diabetes mellitus and isthe most common cause of blindness in working age population. As thedisease progresses, retinal vascular obliteration and hypoxia inducesVEGF upregulation and cause retinal neovascularization which is thehallmark of proliferative diabetic retinopathy. Retinalneovascularization usually causes vitreous hemorrhage and retinaldetachment, which severely impairs vision. Suppression of VEGF canregress the retinal neovascularization in proliferative diabeticretinopathy. In addition, the high intraocular VEGF concentration caninduce vascular leakage and macular edema. Macular edema can impaircentral vision in patients with diabetic retinopathy and other anti-VEGFagents such as bevacizumab, ranibizumab and aflibercept are known to beeffective in the treatment of macular edema.

Retinal Vein Occlusion

There are two types of retinal vein occlusion: branch retinal veinocclusion and central retinal vein occlusion. Retinal vein occlusion isaffected by similar pathogenic mechanism as with diabetic retinopathy:retinal capillary occlusion, hypoxia, and VEGF up-regulation. Thus,retinal neovascularization and macular edema often develop in thiscondition. Other anti-VEGF agents are also known to be effective intreating macular edema from retinal vein occlusion.

Retinopathy of Prematurity

Retinopathy of prematurity is the most common cause of blindness inchildren.

Retinopathy of prematurity is characterized with abnormal retinalneovascularization, which causes tractional detachment of the retina. Atraditional treatment method is laser photocoagulation on the ischemicretina to suppress VEGF and abnormal neovascularization. Recently,down-regulation of VEGF using bevacizumab was shown to be effective inregressing neovascularization and in preventing blindness.

Neovascular Glaucoma

Neovascular glaucoma is a blinding complication of ischemic retinopathysuch as diabetic retinopathy, retinal vein occlusion and ocular ischemicsyndrome. In chronic ocular ischemia, new vessel develops in the irisand in the angle, which blocks the trabecular meshwork and ocularhypertension develops. High intraocular pressure compresses optic nervehead and permanent blindness may develop. This series of pathogenicevents leads to neovascular glaucoma. Anti-VEGF agents have been knownto be efficacious in the prevention and treatment of neovascularglaucoma.

Sequence Listing Free Text

As regards the use of nucleotide symbols other than a, g, c, t, theyfollow the convention set forth in WIPO Standard ST.25, Appendix 2,Table 1, wherein k represents t or g; n represents a, c, t or g; mrepresents a or c; r represents a or g; s represents c or g; wrepresents a or t and y represents c or t.

Table 1 shows SEQ ID NO:1 nucleic acid sequence and its correspondingamino acid sequence (SEQ ID NO:2) for subdomain assemblies of VEGF-Grabbackbone sequence composed of hVEGFR1 signal sequence, VEGFR1 domain 2,VEGFR1 domain 3, and hFC domain portion in order.

Table 2 shows SEQ ID NO:3 nucleic acid sequence and its correspondingamino acid sequence (SEQ ID NO:4) for subdomain assemblies of VEGF-Grab1composed of hVEGFR1 signal sequence, VEGFR1 domain 2, VEGFR1 domain 3,and hFC domain portion in order with the following mutations in VEGFR1domain 3: at nucleic acid positions 403˜405 (amino acid position135Ser), and nucleic acid positions 412˜414 (amino acid position138Thr).

Table 3 shows SEQ ID NO:5 nucleic acid sequence and its correspondingamino acid sequence (SEQ ID NO:6) for subdomain assemblies of VEGF-Grab2composed of hVEGFR1 signal sequence, VEGFR1 domain 2, VEGFR1 domain 3,and hFC domain portion in order with the following mutations in VEGFR1domain 3: at nucleic acid positions 514˜516 (amino acid position172Asn).

Table 4 shows SEQ ID NO:7 nucleic acid sequence and its correspondingamino acid sequence (SEQ ID NO:8) for subdomain assemblies of VEGF-Grab3composed of hVEGFR1 signal sequence, VEGFR1 domain 2, VEGFR1 domain 3,and hFC domain portion in order with the following mutations in VEGFR1domain 3: at nucleic acid positions 403˜405 (amino acid position135Ser), nucleic acid positions 412˜414 (amino acid position 138Thr),and nucleic acid positions 514˜516 (amino acid position 172Asn).

Nucleic Acid Constructs

Also provided is an expression vector comprising a nucleic acid moleculeof the invention as described herein, wherein the nucleic acid moleculeis operatively linked to an expression control sequence. Also providedis a host-vector system for the production of a fusion polypeptide whichcomprises the expression vector of the invention which has beenintroduced into a host cell suitable for expression of the fusionpolypeptide. The suitable host cell may be a bacterial cell such as E.coli, a yeast cell, such as Pichia pastoris, an insect cell, such asSpodoptera frugiperda, or a mammalian cell, such as a COS, HEK or CHOcell.

The present invention also provides for methods of producing the fusionpolypeptides of the invention by growing cells of the host-vector systemdescribed herein, under conditions permitting production of the fusionpolypeptide and recovering the fusion polypeptide so produced. Thefusion polypeptides useful for practicing the present invention may beprepared by expression in a prokaryotic or eukaryotic expression system.

The recombinant gene may be expressed and the polypeptide purifiedutilizing any number of methods. The gene may be subcloned into abacterial expression vector, such as for example, but not by way oflimitation, pZErO.

The fusion polypeptides may be purified by any technique which allowsfor the subsequent formation of a stable, biologically active protein.For example, and not by way of limitation, the factors may be recoveredfrom cells either as soluble proteins or as inclusion bodies, from whichthey may be extracted quantitatively by 8M guanidinium hydrochloride anddialysis. In order to further purify the factors, any number ofpurification methods may be used, including but not limited toconventional ion exchange chromatography, affinity chromatography,different sugar chromatography, hydrophobic interaction chromatography,reverse phase chromatography or gel filtration.

When used herein, fusion polypeptide includes functionally equivalentmolecules in which amino acid residues are substituted for residueswithin the sequence resulting in a silent or conservative change. Forexample, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity, which acts as afunctional equivalent, resulting in a silent or conservative alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. The potential glycosylation amino acids include serine,threonine, and asparagine. Also included within the scope of theinvention are proteins or fragments or derivatives thereof which exhibitthe same or similar biological activity and derivatives which aredifferentially modified during or after translation, e.g., byglycosylation, proteolytic cleavage, linkage to an antibody molecule orother cellular ligand, etc.

Cells that express the fusion polypeptides of the invention aregenetically engineered to produce them by, for example, transfection,transduction, electroporation, or microinjection techniques.

In addition, the present invention contemplates use of the fusionpolypeptides described herein in tagged form.

Any of the methods known to one skilled in the art for the insertion ofDNA fragments into a vector may be used to construct expression vectorsencoding the fusion polypeptides of the invention using appropriatetranscriptional/translational control signals and protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinations (genetic recombination).Expression of nucleic acid sequence encoding the fusion polypeptides ofthe invention may be regulated by a second nucleic acid sequence so thatthe fusion polypeptide is expressed in a host transformed with therecombinant DNA molecule. For example, expression of the fusionpolypeptides described herein may be controlled by any promoter/enhancerelement known in the art. Promoters which may be used to controlexpression of the fusion polypeptide include, but are not limited to thelong terminal repeat as described in Squinto et al., (1991, Cell65:1-20); the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290:304-310), the CMV promoter, the M-MuLV 5′ terminal repeat thepromoter contained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinasepromoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:144-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the β-lactamase promoter (Villa-Kamaroff, et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter(DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25), see also“Useful proteins from recombinant bacteria” in Scientific American,1980, 242:74-94; promoter elements from yeast or other fungi such as theGal 4 promoter, the ADH (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter, andthe following animal transcriptional control regions, which exhibittissue specificity and have been utilized in transgenic animals:elastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology7:425-515); insulin gene control region which is active in pancreaticbeta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin genecontrol region which is active in lymphoid cells (Grosschedl et al.,1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumingene control region which is active in liver (Pinkert et al., 1987,Genes and Devel. 1:268-276), alpha-fetoprotein gene control region whichis active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648;Hammer et al., 1987, Science 235:53-58); alpha 1-antitrypsin genecontrol region which is active in the liver (Kelsey et al., 1987, Genesand Devel. 1:161-171), beta-globin gene control region which is activein myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias etal., 1986, Cell 46:89-94); myelin basic protein gene control regionwhich is active in oligodendrocyte cells in the brain (Readhead et al.,1987, Cell 48:703-712); myosin light chain-2 gene control region whichis active in skeletal muscle (Shani, 1985, Nature 314:283-286), andgonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Thus, according to the invention, expression vectors capable of beingreplicated in a bacterial or eukaryotic host comprising nucleic acidsencoding a fusion polypeptide as described herein, are used to transfectthe host and thereby direct expression of such nucleic acid to producefusion polypeptides which may then be recovered in biologically activeform. As used herein, a biologically active form includes a form capableof binding to the relevant receptor and causing a differentiatedfunction and/or influencing the phenotype of the cell expressing thereceptor.

Expression vectors containing the nucleic acid inserts can be identifiedby without limitation, at least three general approaches: (a) DNA-DNAhybridization, (b) presence or absence of “marker” gene functions, and(c) expression of inserted sequences. In the first approach, thepresence of foreign nucleic acids inserted in an expression vector canbe detected by DNA-DNA hybridization using probes comprising sequencesthat are homologous to an inserted nucleic acid sequences. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of foreign nucleic acid sequences in the vector.For example, if an ell nucleic acid sequence is inserted within themarker gene sequence of the vector, recombinants containing the insertcan be identified by the absence of the marker gene function. In thethird approach, recombinant expression vectors can be identified byassaying the foreign nucleic acid product expressed by the recombinantconstructs. Such assays can be based, for example, on the physical orfunctional properties of the nucleic acid product of interest, forexample, by binding of a ligand to a receptor or portion thereof whichmay be tagged with, for example, a detectable antibody or portionthereof or binding to antibodies produced against the protein ofinterest or a portion thereof

The fusion polypeptide, in particular modified of the present invention,may be expressed in the host cells transiently, constitutively orpermanently.

The invention herein further provides for the development of a fusionpolypeptide as a therapeutic agent for the treatment of patientssuffering from disorders involving cells, tissues or organs whichexpress the VEGF-A, VEGF-B and/or PIGF. Such molecules may be used in amethod of treatment of the human or animal body, or in a method ofdiagnosis.

Effective doses useful for treating these or other diseases or disordersmay be determined using methods known to one skilled in the art (see,for example, Fingl, et al., The Pharmacological Basis of Therapeutics,Goodman and Gilman, eds. Macmillan Publishing Co, New York, pp. 1-46(1975). Pharmaceutical compositions for use according to the inventioninclude the fusion polypeptides described above in a pharmacologicallyacceptable liquid, solid or semi-solid carrier, linked to a carrier ortargeting molecule (e.g., antibody, hormone, growth factor, etc.) and/orincorporated into liposomes, microcapsules, and controlled releasepreparation prior to administration in vivo. For example, thepharmaceutical composition may comprise a fusion polypeptide in anaqueous solution, such as sterile water, saline, phosphate buffer ordextrose solution. Alternatively, the active agents may be comprised ina solid (e.g. wax) or semi-solid (e.g. gelatinous) formulation that maybe implanted into a patient in need of such treatment. Theadministration route may be any mode of administration known in the art,including but not limited to intravenously, intrathecally,subcutaneously, intrauterinely, by injection into involved tissue,intraarterially, intranasally, orally, or via an implanted device.

Administration may result in the distribution of the active agent of theinvention throughout the body or in a localized area. For example, insome conditions, which involve distant regions of the nervous system,intravenous or intrathecal administration of agent may be desirable. Insome situations, an implant containing active agent may be placed in ornear the lesioned area. Suitable implants include, but are not limitedto, gelfoam, wax, spray, or microparticle-based implants.

The present invention also provides for pharmaceutical compositionscomprising the fusion polypeptides described herein, in apharmacologically acceptable vehicle. The compositions may beadministered systemically or locally. Any appropriate mode ofadministration known in the art may be used, including, but not limitedto, intravenous, intrathecal, intraarterial, intranasal, oral,subcutaneous, intraperitoneal, or by local injection or surgicalimplant. Sustained release formulations are also provided for.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingthe inventive chimeric molecule, by way of gene therapy to inhibitangiogenesis. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, nucleic acid sequences may encode VEGF-Grab, inwhich the nucleic acid sequences are part of expression vectors thatexpress the polypeptides in a suitable host. In particular, such nucleicacid sequences have promoters operably linked to the polypeptide codingregion, said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the polypeptide coding sequences and anyother desired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the antibody encoding nucleic acids(Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989);Zijlstra et al., Nature 342:435-438 (1989).

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors, or by direct injection of naked DNA,or coating with lipids or cell-surface receptors or transfecting agents,encapsulation in liposomes, microparticles, or microcapsules, or byadministering them in linkage to a peptide which is known to enter thenucleus, by administering it in linkage to a ligand subject toreceptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.262:4429-4432 (1987)) (which can be used to target cell typesspecifically expressing the receptors) and so on. In another embodiment,nucleic acid-ligand complexes can be formed in which the ligandcomprises a fusogenic viral peptide to disrupt endosomes, allowing thenucleic acid to avoid lysosomal degradation. In yet another embodiment,the nucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor. Alternatively, the nucleicacid can be introduced intracellularly and incorporated within host cellDNA for expression, by homologous recombination (Koller and Smithies,Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding the polypeptide are used. The nucleic acid sequencesencoding the polypeptide to be used in gene therapy are cloned into oneor more vectors, which facilitates delivery of the gene into a patient.Retroviral vectors, adenoviral vectors and adeno-associated viruses areexamples of viral vectors that may be used. Retroviral vectors containthe components necessary for the correct packaging of the viral genomeand integration into the host cell DNA.

Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia because they naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. In addition, adeno-associatedvirus (AAV) has also been proposed for use in gene therapy.

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion andso on. Numerous techniques are known in the art for the introduction offoreign genes into cells and may be used in accordance with the presentinvention, provided that the necessary developmental and physiologicalfunctions of the recipient cells are not disrupted. The technique shouldprovide for the stable transfer of the nucleic acid to the cell, so thatthe nucleic acid is expressible by the cell and preferably heritable andexpressible by its cell progeny.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asT-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, and so on.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding the polypeptide are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention.

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Therapeutic Composition

In one embodiment, the present invention relates to treatment forvarious diseases that are characterized by unwanted blood vesselformation. In this way, the inventive therapeutic compound may beadministered to human patients who are either suffering from, or proneto suffer from the disease by providing a molecule that bind to VEGF-A,VEGF-B and/or PIGF.

The formulation of therapeutic compounds is generally known in the artand reference can conveniently be made to Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example,from about 0.05 ng to about 20 mg per kilogram of body weight per daymay be administered. Dosage regime may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. The activecompound may be administered in a convenient manner such as by the oral,intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intra ocular, intradermal or suppository routes or implanting (egusing slow release molecules by the intraperitoneal route or by usingcells e.g. monocytes or dendrite cells sensitised in vitro andadoptively transferred to the recipient). Depending on the route ofadministration, the peptide may be required to be coated in a materialto protect it from the action of enzymes, acids and other naturalconditions which may inactivate said ingredients.

For example, the low lipophilicity of the peptides will allow them to bedestroyed in the gastrointestinal tract by enzymes capable of cleavingpeptide bonds and in the stomach by acid hydrolysis. In order toadminister peptides by other than parenteral administration, they willbe coated by, or administered with, a material to prevent itsinactivation. For example, peptides may be administered in an adjuvant,co-administered with enzyme inhibitors or in liposomes. Adjuvantscontemplated herein include resorcinols, non-ionic surfactants such aspolyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes.

The active compounds may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propylene glycoland liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsuperfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, chlorobutanol, phenol, sorbic acid, theomersal and the like. Inmany cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecomposition of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterile active ingredient into a sterile vehicle which containsthe basic dispersion medium and the required other ingredients fromthose enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze-drying technique whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

When the peptides are suitably protected as described above, the activecompound may be orally administered, for example, with an inert diluentor with an assimilable edible carrier, or it may be enclosed in hard orsoft shell gelatin capsule, or it may be compressed into tablets, or itmay be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compound may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 5 toabout 80% of the weight of the unit. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosagewill be obtained. Preferred compositions or preparations according tothe present invention are prepared so that an oral dosage unit formcontains between about 0.1 μg and 2000 mg of active compound.

The tablets, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

Delivery Systems

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis, construction of a nucleicacid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, intra ocular,epidural, and oral routes. The compounds or compositions may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compounds or compositions ofthe invention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody or a peptide of the invention, care must be taken to usematerials to which the protein does not absorb. In another embodiment,the compound or composition can be delivered in a vesicle, in particulara liposome. In yet another embodiment, the compound or composition canbe delivered in a controlled release system. In one embodiment, a pumpmay be used. In another embodiment, polymeric materials can be used. Inyet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, thus requiring only a fraction ofthe systemic dose.

Labels

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Glucose oxidase is particularly preferred as it has goodstability and its substrate (glucose) is readily available. Activity ofan oxidase label may be assayed by measuring the concentration ofhydrogen peroxide formed by the enzyme-labeled antibody/substratereaction. Besides enzymes, other suitable labels include radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³H),indium (¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, suchas fluorescein and rhodamine, and biotin.

Examples of suitable enzyme labels include malate dehydrogenase,δ-5-steroid isomerase, yeast-alcohol dehydrogenase, α-glycerol phosphatedehydrogenase, triose phosphate isomerase, peroxidase, alkalinephosphatase, asparaginase, glucose oxidase, β-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is preferred isotope where in vivoimaging is used since its avoids the problem of dehalogenation of the¹²⁵I or ¹³¹I-labeled polypeptide by the liver. In addition, thisradionucleotide has a more favorable gamma emission energy for imaging.For example, ¹¹¹In coupled to monoclonal antibodies with1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in non-tumorstissues, particularly the liver, and therefore enhances specificity oftumor localization.

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, and a fluorescamine label.

Examples of suitable toxin labels include, Pseudomonas toxin, diphtheriatoxin, ricin, and cholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and iron. Deuterium may also be used. Othercontrasting agents also exist for EPR, PET or other imaging mechanisms,which are known to persons of skill in the art.

Typical techniques for binding the above-described labels topolypeptides are provided by Kennedy et al. (Clin. Chim. Acta 70:1-311976 and Schurs et al. Clin. Chim. Acta 81:1-40, 1977). Couplingtechniques include the glutaraldehyde method, the periodate method, thedimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide estermethod, all of which methods are incorporated by reference herein.

The polypeptides and antibodies of the present invention, includingfragments thereof, may be used to detect VEGF-Grab/ligand complex usingbiochip and biosensor technology. Biochip and biosensors of the presentinvention may also comprise antibodies, which specifically recognize thepolypeptides of the present invention to detect VEGF-Grab/ligandcomplex.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. The following examples are offered by way ofillustration of the present invention, and not by way of limitation.

EXAMPLES Example 1 Materials and Methods Example 1.1 Cell Lines

All cell lines for this studies (HUVEC (CC-2519, Lonza), dhfr-deficientCHO cells (CRL-9096, ATCC) and LLC cells (CRL-1642, ATCC)) have not beencultured for longer than 6 months.

Example 1.2 Generation of Recombinant Proteins

Human VEGFR1 (amino acid residues 132-331) fused to human Fc domain ofIgG1 (referred to as Fc) was cloned into the pCMV-dhfr vector. A seriesof VEGF-Grabs were generated by site directed mutagenesis. VEGF-Grabsconstructs were transfected into dhfr-deficient CHO cells. Transfectedcells were selected by G418, and genes were amplified by graduallyincreasing methotrexate treatment (0.001-0.5 μM). The cells were thengrown in HyQSFM4CHO (ThermoScientific) media containing 0.5 μMmethotrexate. VEGF-Grabs and VEGF-Trap were purified by proteinA-sepharose affinity chromatography. Purified proteins were quantitatedusing Bradford assay and confirmed by Coomassie blue staining afterSDS-PAGE.

Example 1.3 Isoelectric Focusing Analysis

To analyze isoelectric points of VEGF Grabs (G1 and G3) and VEGF Trap(VT), 5 μg of proteins and IEF marker (Novex) were loaded on IEF gelsranging pH 3-10 (Novex) and run at 100V for 1 hr, 200V for 1 hr and 500Vfor 30 min. After electrophoresis, gels were stained with silverstaining kit (Elpis).

Example 1.4 In Vitro ECM Binding Assay

Serially diluted G1, G3 or VT (5-80 nM) were treated to the ECM-coatedplate (Becton Dickinson). After washing, HRP conjugated goat anti-humanFc antibody was added and TMB solution was added. The absorbance wasmeasured by ELISA reader at 450 nm.

Example 1.5 In Vivo ECM Binding Assay

100 nM of VR1-Fc, VT, or G3 were treated to the tumor sections whichwere not treated with any protein therapeutics, and the nonspecificallybound VR1-Fc, VT, or G3 were detected with an anti-human Fc-cy3antibody. As an ECM marker, collagen type IV that is a collagenprimarily found in basal lamina of ECM was counterstained with FITC.

Example 1.6 Solid Phase Binding Assay

The binding affinities of VEGF-Grabs to ligands were measured by ELISAas described previously. Briefly, the MaxiSorp plates (Nunc) were coatedwith either hVEGF-A₁₆₅(150 ng/ml), PlGF (62.5 ng/ml), or hVEGF-B (125ng/ml) and serially increasing amounts (0.1 nM-10 μM) of VEGF-Grabs orVEGF-Trap were added. After washing, the plates were incubated withHRP-conjugated goat anti-human Fc antibody. Then,3,3′,5,5′-tetramethylbenzidine (TMB) solution (Sigma-Aldrich) was addedand absorbance was measured by ELISA reader (BioRad) at 450 nm.

Example 1.7 Generation of De-N-Glycosylated Peptides

Glycoproteins were thermally denatured, followed by reduction andalkylation using dithiothreitol and iodoacetamide, respectively. Trypsin(Promega, Madison, Wis.) was added at an enzyme-to-protein ratio of 1:50(w/w) and the mixture was incubated at 37° C. for 16 h.Peptides/glycopeptides were enriched by a C18 peptide trap (Michrom,Auburn, Calif.) and dried by speedvac. N-glycopeptides weredeglycosylated by incubation with 2 μL, peptide N-glycosidase F (NewEngland Biolabs, Ipswich, Mass.) at 37° C. for 16 h. Peptides wereenriched by a C18 peptide trap (Michrom, Auburn, Calif.) and dried byspeedvac.

Example 1.8 Enzymatic Release of N-Glycans

N-glycan release and associated processing steps were carried out aspreviously described. Briefly, glycoproteins were thermally denaturedand reduced in an aqueous solution of ammonium bicarbonate anddithiothreitol prior to digestion by peptide N-glycosidase F at 37° C.for 16 h.

Example 1.9 Enrichment of Glycans and Short Glycopeptides withGraphitized Carbon SPE

Released glycans and glycopeptides were purified by graphitized carbonsolid-phase extraction according to previously optimized procedures.Briefly, graphitized carbon cartridges (Grace Davison, Deerfield, Ill.)were conditioned with water; loaded with aqueous N-glycan solutions; andwashed with water. N-glycans were eluted stepwise with 20% acetonitrilein water, followed by 40% acetonitrile and 0.05% trifluoroacetic acid(v/v) in water. Samples were dried by speedvac.

Example 1.10 Immunoblotting for Detecting VEGFR2 Activation

When HUVECs grown in EGM-2 (Lonza) became confluent, cells were starvedovernight in OPTI-MEM (Invitrogen). VEGF-Grabs or VEGF-Trap (2 μg/ml, 14nM) was then treated for 15 min followed by VEGF-A treatment (50 ng/ml,1 nM) for 10 min. After the treatment, cells were washed with 1×PBS andlysed in lysis buffer. Then, 50 μg of total proteins were loaded on 10%SDS-PAGE, transferred onto nitrocellulose membrane, and immunoblottedwith anti-phospho-VEGFR2 and anti-phospho-ERK1/2 antibody (Cellsignaling). After stripping, VEGFR2 and ERK1/2 were also immunoblotted.

Example 1.11 Migration Assay

HUVECs were seeded on the culture-insert of μ-dish (Ibidi). When cellsbecame confluent, the culture-inserts were removed. Then migrated cellswithin the wound were monitored for 12 hr in the presence of VEGF-A (50ng/ml, 1 nM) and indicated proteins (2 μg/ml, 14 nM) in EBM-2 (Lonza).

Example 1.12 Tube Formation Assay

Matrigel with reduced growth factor (BD Biosciences) was coated onto24-well plate. HUVECs were seeded (5×10⁴ cells per well) and treatedwith VEGF-Grabs or VEGF-Trap (2 μg/ml, 14 nM). After 15 min, VEGF-A (50ng/ml, 1 nM) was added and incubated for 12 hrs. The images were takenunder a microscope.

Example 1.13 Cell Survival Assay

When the seeded HUVECs became confluent, cells were starved in OPTI-MEM(Invitrogen) concurrently treated with VEGF-Grabs or VEGF-Trap (0.35,0.7, 3.5, 7, 35, and 70 nM) in the presence or absence of VEGFA (0.2nM). After 36 hr, WST-1 (water-soluble tetrazolium salt, DOGEN) wasadded and absorbance was measured at 450 nm.

Example 1.14 Migration Assay

Seventy μl of HUVECs at a density of 5×10⁵ cells/ml were seeded on theculture-insert of μ-dish (Ibidi). When cells became confluent, theculture-inserts were removed. Then migrated cells within the wound weremonitored for 12 hr in the presence of VEGF-A (50 ng/ml) and indicatedproteins (2 μg/ml) in EBM-2 (Lonza). The images were taken under amicroscope.

Example 1.15 Mice

Specific pathogen-free (SPF) male C57BL/6J and female MMTV-PyMTtransgenic mice (FVB/N) were purchased from Jackson Laboratory. All micewere anesthetized with 80 mg/kg of ketamine and 12 mg/kg of xylazine,before sacrifice. Particularly for ocular experiments, a total of 15Male C57BL/6J mice, six-week-old, weighing 18-20 g were used. All micewere treated in accordance with the Association for Research in Visionand Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmicand Vision Research. All animal care and experimental procedures wereperformed under the approval (KA2013-42) from the Animal Care Committeeof KAIST.

Example 1.16 PK Analysis

C57BL/6J mice (˜25 g) were given subcutaneous injections of 4, 10, or 25mg/kg VEGF-Grabs and VEGF-Trap. Blood samples were collected from tailsat 1, 2, 4, 8, 12, 24, 48, 96 and 144 hr after injection. The proteinlevels in serum were measured with ELISA.

Example 1.17 Tumor Models and Treatment Regimes

Murine Lewis lung carcinoma (LLC) cells (1×10⁶ cells in 100 μl) weresubcutaneously injected into the dorsal flank of mice (8 to 10-weeksold). Tumor volume was calculated according to the formula:0.5×length×width. VEGF-Grab3 or VEGF-trap (indicated dose) wasintraperitoneally injected at given time points. For the combinedtherapy of G3 with chemotherapeutics, cisplatin (10 mg/kg,Sigma-Aldrich) was intraperitoneally injected at day 9 into LLCtumor-bearing mice that were receiving injections of either VT, or G3.Female MMTV-PyMT mice (12-weeks old) received IP-injections of eitherVT, or G3 (25 mg/kg) twice per week for 3 weeks to test its anti-cancereffects in spontaneous breast cancer model. Mice were anesthetized andtheir primary tumors, LNs, and organs were harvested for histologicalanalyses. Animal care and experimental procedures were performed underthe approval (KA2013-42) from the Animal Care Committee of KAIST.

Example 1.18 Quantitative Real-Time PCR

Total RNA was extracted from the tumor samples using RNeasy plus minikit (Qiagen) followed by cDNA synthesis with SuperScriptII reversetranscriptase (Invitrogen). Quantitative real-time PCR was performedwith indicated primer pairs (Table 5 for the primer sequences) by usingTOPrea1™ qPCR SYBR premix (Enzynomics) and CFX96 real-time PCR detectionsystem (Bio-Rad). The results of the real-time PCR were analysed withCFX manager software (Bio-Rad).

Example 1.19 Histological Analyses

Tumors were processed and stained as previously described. Frozensamples were sectioned and then stained with antibodies (See Table 6 fordetailed information of antibodies). For the visualization of hypoxicareas in the tumors, Hypoxyprobe-1™ (60 mg/kg, solid pimonidazolehydrochloride, Hypoxyprobe) was intravenously injected 90 min beforesacrifice. The tumors were harvested, sectioned, and stained withFITC-conjugated anti-Hypoxyprobe antibody. The CD11b⁺, cytokeratin⁺, andcaspase3⁺ areas were calculated as a percentage per total sectionalarea.

Example 1.20 Ocular Treatment Procedure

C57BL/6J mice subjected to diode 532 nm laser treatment (Lumenis Inc.,Santa Clara, Calif.) after general anesthesia. After a drop of 0.5%tropicamide and 0.5% phenylephrine (Mydrin-P®; Santen Pharmaceutical,Osaka, Japan) is instilled in each eye of each mouse to dilate thepupils, CNV was induced by rupturing the RPE and the underlying Bruch'smembrane. Disruption of Bruch's membrane was induced using a power of100 mW, a spot size of 75 μm and duration of 100 ms to 2.0 times of discdiameters apart from the optic disc. For CNV suppression analysis, themice were then randomly divided into three groups: the VEGF-Trap-treatedgroup that was intravitreally administered commercially availabledrug—Eylea® (Aflibercept) (concentration, 25 mg/ml; dose, 2 μl)Aflibercept is dissolved in an isosmotic liquid (10 mM sodium phosphate,40 mM sodium chloride, 0.03% polysorbate 20, and 5% sucrose [pH 6.2]);the VEGF-Grab3-treated group that was intravitreally administeredVEGF-Grab3 (concentration, 25 mg/ml; dose, 2 μl); and the control groupthat was treated with phosphate-buffered saline (PBS) (2 μl). OIR modelwas established in the mouse by oxygen induction as describedpreviously. Briefly, litters of 7-day-old [postnatal day 7 (P7)]C57BL/6J mice neonates and their mothers were placed in a closedhigh-oxygen chamber for 5 days. An oxygen concentration of 75% wasmaintained and the mice were fed a standard mouse diet and water adlibitum. Then, pups and their mothers were removed at P12 returned toroom air (normoxia) conditions. For OIR regression analysis, the micewere then randomly divided into three groups: the VEGF-Trap-treatedgroup that was intravitreally administered commercially availabledrug—Eylea® (Aflibercept) (concentration, 25 mg/ml; dose, 1 μl); theVEGF-Grab3-treated group that was intravitreally administered VEGF-Grab3(concentration, 25 mg/ml; dose, 1 μl); and the control group that wastreated with phosphate-buffered saline (PBS) (1 μl). At P17, the micewere euthanized for analyses.

Example 1.21 Ocular Sample Excision and Processing

Two weeks after laser photocoagulation for CNV model and 17 days afterOIR pups were born, all mice were anesthetized and perfused through theleft ventricle with 1 mL PBS containing 25 mg/mL of fluoresceinisothiocyanate (FITC)-labeled dextran (2×10⁶ average molecular weight,Sigma, St. Louis, Mo.). After sacrifice of the mouse by cervicaldislocation, the eyes were enucleated and fixed in 2% paraformaldehyde(PFA) in PBS for 5 minutes at room temperature. Under a dissectingmicroscope the cornea, lens and vitreous were removed. The open eyecupwas inserted in a 1% PFA solution for 20 minutes. The remainingretina/choroid/sclera complex was placed on the glass slide. Four radialincisions in the retina/choroid/sclera eyecup were made to prepare aflat mount. The retina was gently separated from the underlyingRPE/choroid/sclera and separately flat mounted. In order to evaluate thechoroidal and retinal vasculature, incubation with hamsteranti-PECAM-1/CD31 (hamster anti-mouse, clone 2H8, MAB1398Z, Millipore,1:200) was performed. In short, after blocking with PBS containing 0.4%Triton X-100 (Sigma-Aldrich, St. Louis, Mo.), 5% normal goat serum(DAKO, Hamburg, Germany), and 20% dimethyl sulfoxide (DMSO; Sigma, St.Louis, Mo.), the RPE/choroid/sclera flat-mounts were incubated 2 days at4° C. with hamster anti-PECAM-1/CD31 primary antibody in blockingbuffer. Anti-PECAM-1/CD31 antibody binds to the surface of endothelialcells and selectively labels the murine vasculature. After washing withPBS, flat mounts incubated 2 days at 4° C. with tetramethylrhodamineisothiocyanate (TRITC)-conjugated goat anti-american hamster IgG(Jackson ImmunoResearch Laboratories, Inc., 1:200) as a secondaryantibody in blocking buffer. After washing with PBS, the cell nucleiwere stained with 10 μg/ml 4′6-diamidino-2-phenylindole (DAPI, Sigma).

Example 1.22 Antibodies

Antibodies used in this study are listed in Table 6.

Example 1.23 Imaging and Quantification

The flat mounts were then examined for CNV. Z-stack images were thencaptured using scanning laser confocal microscopy (Zeiss—LSM780, Zeiss,Jena, Germany). The vasculature filled with FITC-labeled dextran stainedgreen and CNV complexes were identified using the red channel(anti-PECAM-1/CD31 antibody conjugated TRITC). Images of CNV inchoroidal flat mounts were digitalized using an image capture program(LSM Image Browser, Zeiss). Anti-PECAM-1/CD31 antibody conjugated TRITCstained red color area confirms the CNV existence. The total area (inμm²) of FITC-labeled dextran stained green color lesion was calculatedand compared using an image analysis program (Image-J software, NIH,USA). Whole retinal tile-scan images of OIR pups' retinal flat mountswere also obtained using scanning laser confocal microscopy. And then,the retinal vascular intensity was calculated using an image analysisprogram (Image-J software, NIH, USA).

Example 1.24 Statistical Analyses

Values are presented as mean±SD. Statistical differences between meanswere determined by independent sample t-test or analysis of variancewith one-way followed by the Student-Newman-Keuls or Bonferroni test.Statistical significance was set at p<0.05.

Example 2 Results Example 2.1 Design of VEGF-Grab and Their EnhancedBinding Affinities for VEGF-A and PlGF

Both VEGFR1 and VEGFR2 have seven immunoglobulin (Ig)-like domains inthe extracellular domain (FIG. 1A). Among them, VEGFR1 D2 is the primarycontributor to VEGF-A and PlGF binding. In addition, residues in VEGFR1D3 also participate in the high affinity binding of VEGF-A and PlGF.Therefore, the VEGFR1 D2-D3 is the minimal required domain to bind bothVEGF-A and PlGF with high affinity. To design a novel VEGF decoyreceptor fusion protein, w e first analysed the model structures ofVEGFR1 D2-D3/VEGF-A complex generated by MODELLER using templatestructures (PDB ID: 1FLT and 2X1X) (FIGS. 1B and C). The electrostaticpotential analysis revealed that VEGFR1 D3 has abundant positivelycharged amino acids (shown in blue), responsible for the high pI of thisdomain compared to VEGFR2 D3 (FIG. 1 B). Using VEGFR1, we designed VEGFdecoy receptor fusion protein, called VEGF-Grab. VEGF-Grab includeshVEGFR1 signal sequence (amino acids from 1 to 26, nucleotides from 1 to78 which is taken from amino acids from 1 to 26 (nucleotides from 1 to78) of the original hVEGFR1), hVEGFR1 D2-D3 domain (amino acids from 27to 229, nucleotide from 79 to 687 which is taken from amino acids from132 to 332 (nucleotides from 394 to 996) of the original hVEGFR1), andFc domain of human IgG (amino acids from 230 to 459, nucleotides from688 to 1377). In an attempt to reduce the net pI of VEGFR1 D3 andimprove in vivo half-life of VEGF decoy receptor fusion protein, wetargeted residues for mutagenesis located on β1-β2 and β3-β4 loops ofVEGFR1-D3 (FIG. 2) because the model structure of the VEGF-A/VEGFR1D2-D3 complex shows that the β1-β2 loop of hVEGFR1 D3 domain (aminoacids from 133 to 144 of SEQ ID NO:2, nucleotides from 397 to 432 of SEQID NO:1, which is taken from amino acids 236 to 247 (nucleotides 706 to741) of the original hVEGFR1) and the β3-β4 loop of hVEGFR1 D3 domain(amino acids from 164 to 174 of SEQ ID NO:2, nucleotides 490 to 522 ofSEQ ID NO:1, which is taken from amino acids from 267 to 277(nucleotides from 799 to 831) of original hVEGFR1) are not involved inligand binding but are located within the flexible loops as well ascontain many positive residues. Thus, it was predicted that chargeconversion mutations on these loops of VEGF-Grab would maintain theirhigh affinities to VEGF-A/PlGF, reduce the net pI of VEGFR1 D3 domainand avoid structural disruption (FIG. 1C). In addition to the chargeconversion mutation, another rationale for the mutagenesis on these loopis the mutation to Serine, Threonine or Asparagine to be a potential O-or N-glycosylation site which can improve protein half-life.

To test these ides, we generated three VEGFR1 variants; VEGF-Grab1,VEGF-Grab2, and VEGF-Grab3 (FIG. 1A). Three positive-chargedresidues—R135, K138 and the R172, within the VEGFR1 D3 loop region weremutated to negative-charged residues (R135S, K138T and R172N) whereglycans could be attached (FIG. 1A and FIG. 10). All VEGF-Grabconstructs consist of VEGFR1 D2-D3 variants fused to Fc (FIG. 1A).Unfortunately, the expression levels of parental VEGFR1-Fc andVEGF-Grab2 in CHO cells were too low, so further analyses were performedonly with VEGF-Grab1 and VEGF-Grab3. All of the proteins—VEGF-Grab1,VEGF-Grab3, and VEGF-Trap (hereafter, abbreviated as G1, G3, and VT,respectively), were produced from CHO cell and purified by ProteinAaffinity chromatography. Purified G1 and G3 showed diffuse band patternsin reduced SDS-PAGE condition (FIG. 1D), which is a typicalcharacteristic of glycosylated proteins. Under non-reduced conditions,VEGF-Grabs displayed a dimeric form due to the disulfide bond in the Fc(FIG. 1D). The in vitro binding affinities of VEGF-Grabs and VT topro-angiogenic ligands—VEGF-A, PlGF, and VEGF-B, showed that G1(K_(D)=7.9×10⁻¹⁰ M) and G3 (K_(D)=5.6×10⁻¹⁰ M) had 1.1 and 1.5-foldhigher affinity to VEGF-A than VT (K_(D)=8.4×10⁻¹⁰ M) (FIG. 1E).However, the binding affinities of G1 and G3 to VEGF-B, which onlyrequires VEGFR1-D2 for binding, were similar to VT (FIG. 1G).Intriguingly, the binding affinities of G1 (K_(D)=2.8×10⁻⁹ M) and G3(K_(D)=6.9×10⁻⁹ M) to PlGF were 18.5 and 6.7-fold more potent than VT(K_(D)=4.6×10⁻⁸ M), respectively (FIG. 1F), suggesting that wesuccessfully generated new glycosylated-VEGF decoy receptor fusionproteins, G1 and G3, which have significantly higher affinities to bothVEGF-A and PlGF.

Example 2.2 Identification of Newly Added O-Glycan on VEGF-Grabs

Because glycosylation can alter the pI of proteins, we measured the pIof the VEGF-Grabs. Due to the diverse composition of attached glycans,VT, G1, and G3 exhibited micro-heterogeneity with diverse isoform on theisoelectric focusing gel (FIG. 3A). Intriguingly, the pIs of G1 and G3were dramatically decreased to 8.0 and 7.4, respectively (FIG. 3B),compared to that of parental VEGFR1-Fc (pI: 9.4). These pI values werecomparable to that of VT (pI: 7.8). After digestion with PNGaseF, themolecular weights of the VEGF-Grabs and VT were decreased (FIG. 2C),indicating the presence of N-linked glycosylation in VT, G1, and G3. Incontrast to VT, even after PNGaseF digestion, G1 and G3 still displayeddiffuse band patterns suggesting their O-linked glycosylation.

To further confirm the presence of N- or O-glycans at the mutated sitesof VEGF-Grabs, w e performed a mass spectrometry for glycosylationmapping. Calculated masses of both deglycosylated and unglycosylatedtryptic peptides that encompass a potential site for N-glycosylation arelisted in FIG. 11A. LC/MS data indicates that the mutated Asn172 on G3was not glycosylated, whereas three original N-glycosylation sites,Asn61, Asn93, and Asn308 (Asn328 for VT), were all fully occupied (FIG.3E and FIG. 10). Next, using Glyco-Analytical Multispecific Proteolysis(glyco-AMP), we determined whether Ser135 and Thr138 of the VEGF-Grabswere O-glycosylated. From the MS/MS spectrum, O-glycopeptide(STPSPV+HexNAc1Hex1NeuAc1) were observed in both G1 and G3 (FIG. 3D andFIG. 12). Typical glycan fragments for HexNAc, NeuAc-H2O, NeuAc,Hex1NeuAc1, and HexNAc1Hex1NeuAc1 confirmed that VEGF-Grabs areO-glycosylated at Ser135. However, we found no evidence ofO-glycosylation at Thr138.

We then analysed the overall N-glycan compositions of G1, G3, and VT.N-glycan profile was quantified by both nano-LC/MS and MALDI-MS. Thisdata indicated that G1 and G3 exhibited a n increase in high mannose(HM) glycosylation, a decreased in fucosylaed, and complex-undecoratedglycans (C/H-Fuc) as compared to VT (FIG. 11B). Particularly, G3displayed increased sialylation compared to G1 leading to G3's lower pIas compared to G1 (FIG. 3A). This mass spectrometry analysis showed thatboth G1 and G3 have only one additional O-glycosylation site (Ser135)among the mutated residues (FIG. 3E and FIGS. 10-12), as well as threeN-glycosylation sites at 61N, and 93N in VEGFR1 D2 and 308N (328N forVT) in the Fc. In addition, G3 was revealed to be more sialylated thanVT or G1.

Example 2.3 VEGF-Grab3 Exhibits Decreased ECM Binding and Enhanced PKProfiles

Proteins with high pI values bind non-specifically to the ECM, resultingin poor PK and bioavailability. We confirmed that the reduced pI valuesof VEGF-Grabs indeed led to the decreased in vitro ECM binding,comparable to that of VT (FIG. 3K). To demonstrate the reduced in vivoECM binding of G3, we stained control tumor sections with 100 nM ofparental VEGFR1-Fc, VT, or G3. While VEGFR1-Fc bound to the tumorsection non-specifically, no VT or G3 were detected in the tumor section(FIG. 13). These data confirmed lower ECM binding of both G3 and VT invivo, suggesting that the charge conversion at the three mutation sitesand additionally added glycans to Ser135 of G3 allowed it to effectivelyovercome the intrinsic problems of VEGFR1-Fc, non-specific binding toECM. To test whether G1 and G3 displayed an improved in vivo half-life,we analysed their PK profiles at 4 mg/kg dose for 4 days. Interestingly,G3 displayed improved PK profiles (area under the curve (AUC): 59.44μg×days/ml) compared to G1 (AUC: 29.08 μg×days/ml) (FIG. 14). Therefore,we chose G3 for further in vivo anti-cancer studies. Then, we evaluatedthe PK profiles of VT and G3 at varying doses (4, 10, 25 mg/kg) for 6days (FIGS. 3 F and G). VT showed an AUC of 37.57, 34.07, and 65.02μg×days/ml, whereas G3 showed an AUC of 64.36, 65.68, and 117.5μg×days/ml after 4, 10, and 25 mg/kg injections, respectively (FIG. 3H).This reflected a 1.7-, 1.9-, 1.8-fold increase over VT, respectively.This enhanced PK profile of G3 also supports low ECM binding of G3 invivo. Moreover, PK profiles demonstrated that VT was mostly eliminatedby 6 days post-injection, while G3 levels at day 6 remained 2-5 foldhigher than VT (FIGS. 3 F and G), indicating that G3 has a prolongedhalf-life in serum. We also examined the accumulated levels of VT and G3in liver, kidney, tumor and urine of LLC tumor-bearing mice 48 hr aftersubcutaneous injections (4 mg/kg). Higher G3 levels were detected inliver, kidney, and particularly in tumor (18.9-fold increase) than forVT (FIG. 2I and FIG. 15). However, the relative amounts of accumulatedG3 in liver and kidney versus tumor were much lower than that of VT(FIG. 3J), suggesting that most G3 accumulate at the tumor site, whereVEGF-A and PlGF are predominantly produced. No G3 or VT were detected inurine under our experimental conditions. Taken together, these findingssuggest that VEGF-Grab3 has lower ECM binding properties and prolongedhalf-life.

Example 2.4 VEGF-Grabs Inhibits EC Survival, Migration, and TubeFormation Via Suppression of the VEGF Signalling Pathway

VEGF-A promotes proliferation, migration, and survival of endothelialcells through VEGFR2 activation. Accordingly, we examined VEGFR2signalling in HUVECs. Both VEGF-Grabs and VT attenuated VEGF-A-inducedphosphorylation of VEGFR2 and its downstream ERK (FIG. 4A-C, and FIG. 16A-C, see FIG. 17 for dose-dependent inhibitions). In addition,VEGF-Grabs inhibited VEGF-A-induced proliferation of HUVECs with a nIC₅₀ (half maximal inhibitory concentration) at 1.7 nM and 2.4 nM whichare 2.8 and 2-fold more efficient than VT (IC₅₀=4.8 nM), respectively(FIG. 4D). Also, VEGF-Grabs and VT strongly suppressed VEGF-A-inducedmigration (FIGS. 4E and F, and FIGS. 16D and E) and tube formation(FIGS. 4G and H, and FIGS. 16F and G) of HUVECs. These results indicatethat VEGF-Grabs and VT inhibit VEGF-A-induced endothelial cellactivation at comparable levels, through VEGF-A sequestration.

Example 2.5 VEGF-Grab3 Displays Enhanced Anti-Tumor Activity

To evaluate the anti-tumor effects of G3, we employed the LLC tumormodel and treated them with either VT or G3 at 25 mg/kg. G3 treatmentresulted in 61% and 71% reduction in tumor volume and weight, while VTtreatment showed 37% and 29% decreases, respectively (FIGS. 5A and C).In addition, intratumoral necrosis was more dramatic in G3-treatedtumors (35%) than VT-treated tumors (21%) (FIGS. 5B and D). Furthermore,G3-treated tumors exhibited superior anti-angiogenic effects in bothperi- and intratumoral regions and anti-metastatic effects compared withVT-treated tumors, even though there was no significant difference inthe lymphatic vascular density (FIG. 5E-I).

Anti-VEGF therapy induces hypoxia which in turn stimulates therecruitment of TAM into intratumoral hypoxic region. TAM usually expressprofound pro-angiogenic and angiogenesis-modulating factors tore-vascularize the tumor. Interestingly, despite such a significantincrease in hypoxia in both VT- and G3-treated tumors (FIGS. 5J and K),the level of macrophage infiltration in the G3-treated group remainednearly the same as that in non-treated control tumor (FIGS. 5L and M),which, we believe, is attributed to the increased affinity of G3 to PlGFcompared with VT. We next compared the various gene expression profilesin tumors. G3 treatment down-regulated pro-angiogenic genes includingVEGF-A, PlGF, VEGF-C, VEGFR1, and VEGFR2. G3 also reduced Bv8 and CCL2expression compared with VT. These two genes are critical in myeloidcell recruitment to the tumor which can subsequently causerefractoriness to anti-angiogenic therapy (FIG. 5N). Furthermore, noobvious differences were observed in H&E stained sections of vitalorgans including heart, lung, liver, and kidney of VT- or G3-treatedmice (FIG. 18). We also assessed the anti-tumor effect of G3 onestablished macroscopic tumors (>500 mm³). These results showed 27% and46% delays in tumor growth after VT and G3 treatment, respectively (FIG.19), implying that G3 is a promising agent even against bulkymacroscopic tumors.

To confirm dose-responsiveness, tumor-bearing mice received injectionsof varying doses (5, 10, 25, and 50 mg/kg) of VT, or G3 every 2 days.The tumor growth of G3-treated group was gradually reduced in adose-dependent manner with the maximal effect at 50 mg/kg. However, nodistinct differences on tumor growth were observed between 25 mg/kg and50 mg/kg in VT-treated group, whose efficiency, strikingly, wascomparable to that of 10 mg/kg treatment of G3 (FIGS. 5O and P). Interms of tumor metastasis, VT and G3 both showed maximal anti-metastaticeffects at 50 mg/kg (FIGS. 5Q and R). No significant changes were foundin kidney or liver stained with H&E for any dosage of VT or G3 (FIG.20). Taken together, these results suggest that the higher affinity toVEGF-A and PlGF and prolonged half-life of G3 contribute to its higherefficacy in cancer treatment over VT.

Combining anti-VEGF therapy with chemotherapeutics is a valuabletherapeutic strategy, as anti-VEGF therapy alone is not sufficient toinduce complete regression of bulky tumors. The combined therapy of G3and cisplatin displayed the most potent anti-tumor effect (78% reductionin tumor volume) in comparison to cisplatin monotherapy (33%) orcombined therapy of VT and cisplatin (57%) (FIG. 5S). In addition,intratumoral apoptosis of tumor cells increased by >2-fold in thecisplatin+G3 combination group compared with cisplatin monotherapy(FIGS. 5T and U, FIG. 21). These results highlight G3 as a potenttherapeutic option for combination chemotherapies.

Example 2.6 VEGF-Grab3 Also Suppresses Tumor Growth, Angiogenesis, andMetastasis in a Spontaneous Breast Cancer Model

To determine whether G3 consistently inhibits tumor progression in othertumor models, we confirmed our findings using a spontaneous breastcancer model—MMTV-PyMT mice. VT- and G3-treatment reduced the averagesize of tumor nodules by 34% and 61% compared to the control,respectively (FIG. 6B). Tumor sections stained with H&E showed thatcontrol MMTV-PyMT tumors displayed solid sheets of invasive tumor cells(Inv) with no remaining mammary gland structure. In contrast, inG3-treated tumor nodules, more early carcinoma lesions (Ea) wereobserved, in which the boundaries (dotted lines) between early carcinomaand surrounding adipose tissue (Adi) were well preserved (FIG. 6A). G3reduced tumor vascular densities by 45% and 53% in peri- andintratumoral regions versus control, respectively, as compared to the27% and 28% decreases observed in VT-treated tumors (FIGS. 6C and D).Tumor cell apoptosis was also significantly increased in G3-treatedtumors compared to controls or VT-treated tumors (FIGS. 6E and F). Themetastatic tumor cells were 64% and 52% less abundant in the axillaryLNs of G3 and VT-treated mice, respectively, compared to the control,whereas we could not identify any significant differences in thelymphatic vascular density (FIG. 6G-I). These findings demonstrate thatthe increased anti-angiogenic activity of G3 effectively suppressestumor growth and metastasis in a breast cancer model as well.

Example 2.7 VEGF-Grab3 Provides Durable Suppression of TumorAngiogenesis

It has been reported that new vascular sprouts begin to regrow fromremaining tumor vasculatures shortly after the cessation ofanti-angiogenic therapy. To examine the durability of G3, we treatedtumor-bearing mice with either VT, or G3 repeatedly at given time points(FIG. 7A, green arrows). We then withdrew treatment and analysed theremodelling of tumor vasculatures at D17 and D19 (FIG. 7A, blue arrows).While the control tumor vessels showed a 17% increase in tumor vasculardensity and a 24% increase in vascular sprouts between D17 (2 daysoff-treatment) and D19 (4 days off-treatment), VT-withdrawn tumorvessels at D19 showed 51% increase in vascular density and a 91%increase in vascular sprouts compared with D17, indicating a vigorousregrowth of tumor vessels upon cessation of conventional anti-VEGFtreatment. In contrast to VT, G3-withdrawn tumor vessels showed only 20%and 22% increase in vascular density and vascular sprouts at D19compared with D17, which are comparable to those of the control (FIG.7B-D). These findings demonstrate a lasting suppressive effect of G3 ontumor angiogenesis compared to VT.

Example 2.8 Intravitreal Injection of VEGF-Grab3 Inhibits ChoroidalNeovascularization and OIR Non-Perfusion Area Regression

To determine whether G3 decreased CNV size, the area of CNV was measuredby immunofluorescence confocal imaging 14 days after the induction ofCNV. G3-treated mice demonstrated a significant regression inestablished CNV compared to PBS-treated control mice. The CNV size ofPBS-treated control mice was 10803.34 μm³ (range from 3017.24 μm³ to38898.73 μm³, n=60) at 14 days after treatment. However, the CNV size ofG3-treated mice significantly decreased to 5193.76 μm³ (range from270.52 μm³ to 11563.26 μm³, n=60) (P<0.01) (FIG. 8A). As shown inrepresentative images of CNVs (FIG. 8B), CNVs size was significantlysuppressed in the case of G3 treatment compared to PBS-treatment.Aflibercept-treated mice also demonstrated a significant CNV regression.CNV size was 5221.77 μm³ (range from 439.68 μm³ to 15984.70 μm³, n=60)after Aflibercept treatment (P<0.01). Compared with the G3-treated mice,there was no significant difference in the CNV size of theAflibercept-treated mice. The size of CNVs area among three groups andtheir statistical significances are shown in FIG. 8B. In the OIR mouse,treatment with VEGF-Grab increased the area of avascular retina at P17compared with the PBS-injected control eyes. There was a near 2-foldincrease in the vessel intensity of PBS injected eyes at P17 comparedwith the G3 treated eyes (P<0.01) (FIG. 9B). As shown in representativemagnified images of retinal vasculature (FIG. 9A), vascular density wassignificantly suppressed in the case of G3 treatment compared toPBS-treatment. Our results demonstrate that one administration of G3could inhibit laser-induced CNV and OIR Non-perfusion Area Regressioneffectively, highlighting G3 as a potential therapeutic option for AMDand DME.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. The following examples are offered by way ofillustration of the present invention, and not by way of limitation.

TABLE 1 VEGF-Grab Backbone10             20             30             40             50             60|              |              |              |              |             |ATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CTG TGC GCG CTG CTC AGC TGT CTG CTT CTCTAC CAG TCG ATG ACC CTG TGG CCC CAG GAC GAC ACG CGC GAC GAG TCG ACA GAC GAA GAGMet Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser Cys Leu Leu Leu1_______________5__________hVEGFR1 SIGNAL SEQUENCE______15__________________2070             80             90             100            110            120|              |              |              |               |              |ACA GGA TCT AGT TCA GGT GAA TTC GGT AGA CCT TTC GTA GAG ATG TAC AGT GAA ATC CCCTGT CCT AGA TCA AGT CCA CTT AAG CCA TCT GGA AAG CAT CTC TAC ATG TCA CTT TAG GGGThr Gly Ser Ser Ser Gly Glu Phe Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro21______________25__26  27__________VEGFR1 DOMAIN 2_______35________________40130            140            150            160            170            180|              |              |              |              |             |GAA ATT ATA CAC ATG ACT GAA GGA AGG GAG CTC GTC ATT CCC TGC CGG GTT ACG TCA CCTCTT TAA TAT GTG TAC TGA CTT CCT TCC CTC GAG CAG TAA GGG ACG GCC CAA TGC AGT GGAGlu Ile Ile His Met Tyr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro41______________45__________________VEGFR1 DOMAIN 2_____55__________________60190            200            210            220            230            240|              |              |              |              |             |AAC ATC ACT GTT ACT TTA AAA AAG TTT CCA CTT GAC ACT TTG ATC CCT GAT GGA AAA CGCTTG TAG TGA CAA TGA AAT TTT TTC AAA GGT GAA CTG TGA AAC TAG GGA CTA CCT TTT GCGAsn Ile Thr Val The Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg61______________65__________________VEGFR1 DOMAIN 2_____75__________________80250            260            270            280            290            300|              |              |              |              |             |ATA ATC TGG GAC AGT AGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA GAA ATA GGGTAT TAG ACC CTG TCA TCT TTC CCG AAG TAG TAT AGT TTA CGT TGC ATG TTT CTT TAT CCCIle Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly81______________85__________________VEGFR1 DOMAIN 2_____95__________________100310            320            330            340            350            360|              |              |              |              |             |CTT CTG ACC TGT GAA GCA ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT CTC ACA CATGAA GAC TGG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TTC TGT TTG ATA GAG TGT GTALeu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His101_________VEGFR1 DOMAIN 2_________110_111 112_______VEGFR1 DOMAIN 3_______120370            380            390            400            410            420|              |              |              |              |             |CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GTC AAA TTA CTTGCT GTT TGG TTA TGT TAG TAT CTA CAG GTT TAT TCG TGT GGT GCG GGT CAG TTT AAT GAAArg Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val Lys Leu Leu121_____________125_________________VEGFR1 DOMAIN 3_____135_________________140430            440            450            460            470            480|              |              |              |              |             |AGA GGC CAT ACT CTT GTC CTC AAT TGT ACT GCT ACC ACT CCC TTG AAC ACG AGA GTT CAATCT CCG GTA TGA GAA CAG GAG TTA ACA TGA CGA TGG TGA GGG AAC TTG TGC TCT CAA GTTArg Gly His Thr Leu Val Leu Asn Cys Thr Arg Thr Thr Pro Leu Asn Thr Arg Val Gln141_____________145_________________VEGFR1 DOMAIN 3_____155_________________160490            500            510            520            530            540|              |              |              |              |             |ATG ACC TGG AGT TAC CCT GAT GAA AAA AAT AAG AGA GCT TCC GTA AGG CGA CGA ATT GACTAC TGG ACC TCA ATG GGA CTA CTT TTT TTA TTC TCT CGA AGG CAT TCC GCT GCT TAA CTGMet Thr Trp Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp161_____________165_________________VEGFR1 DOMAIN 3_____175_________________180550            560            570            580            590            600|              |              |              |              |             |CAA AGC AAT TCC CAT GCC AAC ATA TTC TAC AGT GTT CTT ACT ATT GAC AAA ATG CAG AACGTT TCG TTA AGG GTA CGG TTG TAT AAG ATG TCA CAA GAA TGA TAA CTG TTT TAC GTC TTGGln Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn181_____________185_________________VEGFR1 DOMAIN 3_____195_________________200610            620            630            640            650            660|              |              |              |              |             |AAA GAC AAA GGA CTT TAT ACT TGT CGT GTA AGG AGT GGA CCA TCA TTC AAA TCT GTT AACTTT CTG TTT CCT GAA ATA TGA ACA GCA CAT TCC TCA CCT GGT AGT AAG TTT AGA CAA TTGLys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys Ser Val Asn201_____________205_________________VEGFR1 DOMAIN 3_____215_________________220670            680            690            700            710            720 |              |              |              |              |             |ACC TCA GTG CAT ATA TAT GAT AAA GCA CTC GAG GAC AAA ACT CAC ACA TGC CCA CCG TGCTGG AGT CAC GTA TAT ATA CTA TTT CGT GAG CTC CTG TTT TGA GTG TGT ACG GGT GGC ACGThr Ser Val His Ile Tyr Asp Lys Ala Leu Glu Asp Lys Thr His Thr Cys Pro Pro Cys221_________VEGFR1 DOMAIN 3_____229 230_____________hFC DOMAIN______________240730            740            750            760            770            780|              |              |              |              |             |CCA GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GACGGT CGT GGA CTT GAG GAC CCC CCT GGC AGT CAG AAG GAG AAG GGG GGT TTT GGG TTC CTGPro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp241_____________245_____________________hFC DOMAIN______255_________________260790            800            810            820            830            840|              |              |              |              |             |ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAATGG GAG TAC TAG AGG GCC TGG GGA CTC CAG TGT ACG CAC CAC CAC CTG CAC TCG GTG CTTThr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu261_____________265_____________________hFC DOMAIN______275_________________280850            860            870            880            890            900|              |              |              |              |             |GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACACTG GGA CTC CAG TTC AAG TTG ACC ATG CAC CTG CCG CAC CTC CAC GTA TTA CGG TTC TGTAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr281_____________285_____________________hFC DOMAIN______295_________________300910            920            930            940            950            960|              |              |              |              |             |AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTGTTC GGC GCC CTC CTC GTC ATG TTG TCG TGC ATG GCA CAC CAG TCG CAG GAG TGG CAG GACLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu301_____________305_____________________hFC DOMAIN______315_________________320970            980            990           1000           1010           1020|              |              |             |              |             |CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCAGTG GTC CTG ACC GAC TTA CCG TTC CTC ATG TTC ACG TTC CAG AGG TTG TTT CGG GAG GGTHis Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro321_____________325_____________________hFC DOMAIN______335_________________3401030          1040           1050           1060           1070           1080|             |              |              |              |             |GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TACCGG GGG TAG CTC TTT TGG TAG AGG TTT CGG TTT CCC GTC GGG GCT CTT GGT GTC CAC ATGAla Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr341_____________345_____________hFC DOMAIN______________355_________________3601090          1100           1110           1120           1130           1140|             |              |              |              |             |ACC CTG CCC CCA TCC CGG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTCTGG GAC GGG GGT AGG GCC CTC CTC TAC TGG TTC TTG GTC CAG TCG GAC TGG ACG GAC CAGThr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val361_____________365_____________________hFC DOMAIN______375_________________3801150          1160           1170           1180           1190           1200|             |              |              |              |             |AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AACTTT CCG AAG ATA GGG TCG CTG TAG CGG CAC CTC ACC CTC TCG TTA CCC GTC GGC CTC TTGLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn381_____________385_____________________hFC DOMAIN______395_________________4001210          1220           1230           1240           1250           1260|             |              |              |              |             |AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AAGTTG ATG TTC TGG TGC GGA GGG CAC GAC CTG AGG CTG CCG AGG AAG AAG GAG ATG TCG TTCAsn Tyr Lys Tyr Tyr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys401_____________405_____________________hFC DOMAIN______415_________________4201270          1280           1290           1300           1310           1320|             |              |              |              |             |CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CATGAG TGG CAC CTG TTC TCG TCC ACC GTC GTC CCC TTG CAG AAG AGT ACG AGG CAC TAC GTALys Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His421_____________425_____________________hFC DOMAIN______435_________________4401330          1340           1350           1360           1370           1377|             |              |              |              |             |GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA TGA(SEQ ID NO: 1)CTC CGA GAC GTG TTG GTG ATG TGC GTC TTC TCG GAG AGG GAC AGA GGC CCA TTT ACTGlu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys ***(SEQ ID NO: 2)441_____________445_____________________hFC DOMAIN______455_____________459

TABLE 2 VEGF-Grab1              10             20             30              40             5060             |              |              |                |             |       |ATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CTG TGC GCG CTG CTC AGC TGT CTG CTT CTCTAC CAG TCG ATG ACC CTG TGG CCC CAG GAC GAC ACG CGC GAC GAG TCG ACA GAC GAA GAGMet Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser Cys Leu Leu Leu1_______________5__________hVEGFR1 SIGNAL SEQUENCE______15__________________20            70             80             90             100             110            120             |              |              |               |              |           |ACA GGA TCT AGT TCA GGT GAA TTC GGT AGA CCT TTC GTA GAG ATG TAC AGT GAA ATC CCCTGT CCT AGA TCA AGT CCA CTT AAG CCA TCT GGA AAG CAT CTC TAC ATG TCA CTT TAG GGGThr Gly Ser Ser Ser Gly Glu Phe Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro21______________25__26  27__________VEGFR1 DOMAIN 2_______35________________40            130            140           150             160             170            180             |              |              |               |              |           |GAA ATT ATA CAC ATG ACT GAA GGA AGG GAG CTC GTC ATT CCC TGC CGG GTT ACG TCA CCTCTT TAA TAT GTG TAC TGA CTT CCT TCC CTC GAG CAG TAA GGG ACG GCC CAA TGC AGT GGAGlu Ile Ile His Met Tyr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro41______________45__________________VEGFR1 DOMAIN 2_____55__________________60            190            200           210             220             230            240             |              |              |               |              |           |AAC ATC ACT GTT ACT TTA AAA AAG TTT CCA CTT GAC ACT TTG ATC CCT GAT GGA AAA CGCTTG TAG TGA CAA TGA AAT TTT TTC AAA GGT GAA CTG TGA AAC TAG GGA CTA CCT TTT GCGAsn Ile Thr Val The Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg61______________65__________________VEGFR1 DOMAIN 2_____75__________________80            250            260           270             280             290            300             |              |              |               |              |           |ATA ATC TGG GAC AGT AGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA GAA ATA GGGTAT TAG ACC CTG TCA TCT TTC CCG AAG TAG TAT AGT TTA CGT TGC ATG TTT CTT TAT CCCIle Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly81______________85__________________VEGFR1 DOMAIN 2_____95__________________100            310            320           330             340             350            360             |              |              |               |              |           |  CTT CTG ACC TGT GAA GCA ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT CTC ACA CATGAA GAC TGG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TTC TGT TTG ATA GAG TGT GTALeu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His101_________VEGFR1 DOMAIN 2_________110_111 112_______VEGFR1 DOMAIN 3_______120            370            380           390             400             410             420             |              |              |               |              |           |CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA AGC CCA GTC ACA TTA CTTGCT GTT TGG TTA TGT TAG TAT CTA CAG GTT TAT TCG TGT GGT TCG GGT CAG TGT AAT GAAArg Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Ser Pro Val Thr Leu Leu121_____________125_________________VEGFR1 DOMAIN 3_____135_________________140            430            440           450             460             470            480             |              |              |               |              |           |AGA GGC CAT ACT CTT GTC CTC AAT TGT ACT GCT ACC ACT CCC TTG AAC ACG AGA GTT CAATCT CCG GTA TGA GAA CAG GAG TTA ACA TGA CGA TGG TGA GGG AAC TTG TGC TCT CAA GTTArg Gly His Thr Leu Val Leu Asn Cys Thr Arg Thr Thr Pro Leu Asn Thr Arg Val Gln141_____________145_________________VEGFR1 DOMAIN 3_____155_________________160            490            500           510             520             530            540             |              |              |               |              |           |ATG ACC TGG AGT TAC CCT GAT GAA AAA AAT AAG AGA GCT TCC GTA AGG CGA CGA ATT GACTAC TGG ACC TCA ATG GGA CTA CTT TTT TTA TTC TCT CGA AGG CAT TCC GCT GCT TAA CTGMet Thr Trp Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp161_____________165_________________VEGFR1 DOMAIN 3_____175_________________180            550            560           570             580             590            600             |              |              |              |               |           |CAA AGC AAT TCC CAT GCC AAC ATA TTC TAC AGT GTT CTT ACT ATT GAC AAA ATG CAG AACGTT TCG TTA AGG GTA CGG TTG TAT AAG ATG TCA CAA GAA TGA TAA CTG TTT TAC GTC TTGGln Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn181_____________185_________________VEGFR1 DOMAIN 3_____195_________________200            610            620           630             640             650            660             |              |              |              |               |           |AAA GAC AAA GGA CTT TAT ACT TGT CGT GTA AGG AGT GGA CCA TCA TTC AAA TCT GTT AACTTT CTG TTT CCT GAA ATA TGA ACA GCA CAT TCC TCA CCT GGT AGT AAG TTT AGA CAA TTGLys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys Ser Val Asn201_____________205_________________VEGFR1 DOMAIN 3_____215_________________220            670            680           690             700             710            720             |              |              |              |               |           |ACC TCA GTG CAT ATA TAT GAT AAA GCA CTC GAG GAC AAA ACT CAC ACA TGC CCA CCG TGCTGG AGT CAC GTA TAT ATA CTA TTT CGT GAG CTC CTG TTT TGA GTG TGT ACG GGT GGC ACGThr Ser Val His Ile Tyr Asp Lys Ala Leu Glu Asp Lys Thr His Thr Cys Pro Pro Cys221_________VEGFR1 DOMAIN 3_____229 230_____________hFC DOMAIN______________240            730            740           750             760             770            780             |              |              |              |               |           |CCA GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GACGGT CGT GGA CTT GAG GAC CCC CCT GGC AGT CAG AAG GAG AAG GGG GGT TTT GGG TTC CTGPro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp241_____________245_____________________hFC DOMAIN______255_________________260            790            800           810             820             830            840             |              |              |              |               |           |ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAATGG GAG TAC TAG AGG GCC TGG GGA CTC CAG TGT ACG CAC CAC CAC CTG CAC TCG GTG CTTThr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu261_____________265_____________________hFC DOMAIN _____275_________________280            850            860           870             880             890            900             |              |              |              |               |           |GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACACTG GGA CTC CAG TTC AAG TTG ACC ATG CAC CTG CCG CAC CTC CAC GTA TTA CGG TTC TGTAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr281_____________285_____________________hFC DOMAIN______295_________________300            910            920           930             940             950            960             |              |              |              |               |           |AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTGTTC GGC GCC CTC CTC GTC ATG TTG TCG TGC ATG GCA CAC CAG TCG CAG GAG TGG CAG GACLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu301_____________305_____________________hFC DOMAIN______315_________________320            970           980           990            1000           1010            1020             |              |              |              |               |           |CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCAGTG GTC CTG ACC GAC TTA CCG TTC CTC ATG TTC ACG TTC CAG AGG TTG TTT CGG GAG GGTHis Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro321_____________325_____________________hFC DOMAIN______335_________________340          1030            1040          1050           1060           1070          1080             |              |              |              ||                           |GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TACCGG GGG TAG CTC TTT TGG TAG AGG TTT CGG TTT CCC GTC GGG GCT CTT GGT GTC CAC ATGAla Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr341_____________345_____________hFC DOMAIN______________355_________________360          1090            1100          1110            1120           1130          1140             |              |              |              |               |           |ACC CTG CCC CCA TCC CGG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTCTGG GAC GGG GGT AGG GCC CTC CTC TAC TGG TTC TTG GTC CAG TCG GAC TGG ACG GAC CAGThr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val361_____________365_____________________hFC DOMAIN______375_________________380          1150            1160          1170            1180           1190          1200             |              |              |              |               |           |AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AACTTT CCG AAG ATA GGG TCG CTG TAG CGG CAC CTC ACC CTC TCG TTA CCC GTC GGC CTC TTGLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn381_____________385_____________________hFC DOMAIN______395_________________400          1210            1220          1230            1240           1250          1260             |              |              |              |               |           |AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AAGTTG ATG TTC TGG TGC GGA GGG CAC GAC CTG AGG CTG CCG AGG AAG AAG GAG ATG TCG TTCAsn Tyr Lys Tyr Tyr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys401_____________405_____________________hFC DOMAIN______415_________________420          1270            1280          1290            1300           1310          1320             |              |              |              |               |           |CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CATGAG TGG CAC CTG TTC TCG TCC ACC GTC GTC CCC TTG CAG AAG AGT ACG AGG CAC TAC GTALys Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His421_____________425_____________________hFC DOMAIN______435_________________440          1330            1340          1350            1360           1370          1377             |              |              |              |               |           |GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA TGA(SEQ ID NO: 3)CTC CGA GAC GTG TTG GTG ATG TGC GTC TTC TCG GAG AGG GAC AGA GGC CCA TTT ACTGlu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys ***(SEQ ID NO: 4)441_____________445_____________________hFC DOMAIN______455_____________459

TABLE 3 VEGF-Grab2            10             20             30              40             50            60             |              |              |               |              |           |ATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CTG TGC GCG CTG CTC AGC TGT CTG CTT CTCTAC CAG TCG ATG ACC CTG TGG CCC CAG GAC GAC ACG CGC GAC GAG TCG ACA GAC GAA GAGMet Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser Cys Leu Leu Leu1_______________5__________hVEGFR1 SIGNAL SEQUENCE______15__________________20            70             80             90             100            110            120            |               |              |               |              |           |ACA GGA TCT AGT TCA GGT GAA TTC GGT AGA CCT TTC GTA GAG ATG TAC AGT GAA ATC CCCTGT CCT AGA TCA AGT CCA CTT AAG CCA TCT GGA AAG CAT CTC TAC ATG TCA CTT TAG GGGThr Gly Ser Ser Ser Gly Glu Phe Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro21______________25__26  27__________VEGFR1 DOMAIN 2_______35________________40            130          140             150             160            170            180             |             |               |               |              |           |GAA ATT ATA CAC ATG ACT GAA GGA AGG GAG CTC GTC ATT CCC TGC CGG GTT ACG TCA CCTCTT TAA TAT GTG TAC TGA CTT CCT TCC CTC GAG CAG TAA GGG ACG GCC CAA TGC AGT GGAGlu Ile Ile His Met Tyr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro41______________45__________________VEGFR1 DOMAIN 2_____55__________________60            190          200             210             220            230            240             |             |               |               |              |           |AAC ATC ACT GTT ACT TTA AAA AAG TTT CCA CTT GAC ACT TTG ATC CCT GAT GGA AAA CGCTTG TAG TGA CAA TGA AAT TTT TTC AAA GGT GAA CTG TGA AAC TAG GGA CTA CCT TTT GCGAsn Ile Thr Val The Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg61__________________65______________VEGFR1 DOMAIN 2_____75__________________80            250          260             270             280            290            300             |             |               |               |              |           |ATA ATC TGG GAC AGT AGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA GAA ATA GGGTAT TAG ACC CTG TCA TCT TTC CCG AAG TAG TAT AGT TTA CGT TGC ATG TTT CTT TAT CCCIle Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly81______________85__________________VEGFR1 DOMAIN 2_____95__________________100            310          320             330             340            350            360             |             |               |               |              |           |CTT CTG ACC TGT GAA GCA ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT CTC ACA CATGAA GAC TGG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TTC TGT TTG ATA GAG TGT GTALeu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His101_________VEGFR1 DOMAIN 2_________110_111 112_______VEGFR1 DOMAIN 3_______120             370          380             390             400            410            420             |             |               |               |              |           |CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA CGC CCA GTC AAA TTA CTTGCT GTT TGG TTA TGT TAG TAT CTA CAG GTT TAT TCG TGT GGT GCC GGT CAG TTT AAT GAAArg Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val Lys Leu Leu121_____________125_________________VEGFR1 DOMAIN 3_____135_________________140            430          440             450             460            470            480             |             |               |               |              |           |AGA GGC CAT ACT CTT GTC CTC AAT TGT ACT GCT ACC ACT CCC TTG AAC ACG AGA GTT CAATCT CCG GTA TGA GAA CAG GAG TTA ACA TGA CGA TGG TGA GGG AAC TTG TGC TCT CAA GTTArg Gly His Thr Leu Val Leu Asn Cys Thr Arg Thr Thr Pro Leu Asn Thr Arg Val Gln141_____________145_________________VEGFR1 DOMAIN 3_____155_________________160            490          500             510             520            530            540             |             |               |               |              |           |ATG ACC TGG ACT TAC CCT GAT GAA AAA AAT AAG AAC GCT TCC GTA AGG CGA CGA ATT GACTAC TGG ACC TCA ATG GGA CTA CTT TTT TTA TTC TTG CGA AGG CAT TCC GCT GCT TAA CTGMet Thr Trp Ser Tyr Pro Asp Glu Lys Asn Lys Asn Ala Ser Val Arg Arg Arg Ile Asp161_____________165_________________VEGFR1 DOMAIN 3_____175_________________180            550          560             570             580            590            600             |             |               |               |              |           |CAA AGC AAT TCC CAT GCC AAC ATA TTC TAC AGT GTT CTT ACT ATT GAC AAA ATG CAG AACGTT TCG TTA AGG GTA CGG TTG TAT AAG ATG TCA CAA GAA TGA TAA CTG TTT TAC GTC TTGGln Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn181_____________185_________________VEGFR1 DOMAIN 3_____195_________________200            610          620             630             640            650            660             |             |               |               |              |           |AAA GAC AAA GGA CTT TAT ACT TGT CGT GTA AGG AGT GGA CCA TCA TTC AAA TCT GTT AACTTT CTG TTT CCT GAA ATA TGA ACA GCA CAT TCC TCA CCT GGT AGT AAG TTT AGA CAA TTGLys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys Ser Val Asn201_____________205_________________VEGFR1 DOMAIN 3_____215_________________220            670          680             690             700            710            720             |             |               |               |              |           |ACC TCA GTG CAT ATA TAT GAT AAA GCA CTC GAG GAC AAA ACT CAC ACA TGC CCA CCG TGCTGG AGT CAC GTA TAT ATA CTA TTT CGT GAG CTC CTG TTT TGA GTG TGT ACG GGT GGC ACGThr Ser Val His Ile Tyr Asp Lys Ala Leu Glu Asp Lys Thr His Thr Cys Pro Pro Cys221_________VEGFR1 DOMAIN 3_____229 230_____________hFC DOMAIN______________240            730          740             750             760            770            780             |             |               |               |              |           |CCA GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GACGGT CGT GGA CTT GAG GAC CCC CCT GGC AGT CAG AAG GAG AAG GGG GGT TTT GGG TTC CTGPro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp241_____________245_____________________hFC DOMAIN______255_________________260            790          800             810             820            830            840             |             |               |               |              |           |ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAATGG GAG TAC TAG AGG GCC TGG GGA CTC CAG TGT ACG CAC CAC CAC CTG CAC TCG GTG CTTThr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu261_____________265_____________________hFC DOMAIN______275_________________280            850          860             870             880            890            900             |             |               |               |              |           |GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACACTG GGA CTC CAG TTC AAG TTG ACC ATG CAC CTG CCG CAC CTC CAC GTA TTA CGG TTC TGTAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr281_____________285_____________________hFC DOMAIN______295_________________300            910          920             930             940            950            960             |             |               |               |              |           |AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTGTTC GGC GCC CTC CTC GTC ATG TTG TCG TGC ATG GCA CAC CAG TCG CAG GAG TGG CAG GACLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu301_____________305_____________________hFC DOMAIN______315_________________320            970          980             990            1000           1010            1020              |             |               |               |              |           |CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCAGTG GTC CTG ACC GAC TTA CCG TTC CTC ATG TTC ACG TTC CAG AGG TTG TTT CGG GAG GGTHis Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro321_____________325_____________________hFC DOMAIN______335_________________340            1030        1040            1050            1060            1070            1080             |             |               |               |              |           |GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TACCGG GGG TAG CTC TTT TGG TAG AGG TTT CGG TTT CCC GTC GGG GCT CTT GGT GTC CAC ATGAla Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr341_____________345_____________________hFC DOMAIN______355_________________360            1090        1100            1110            1120            1130            1140             |             |               |              |               |           |ACC CTG CCC CCA TCC CGG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTCTGG GAC GGG GGT AGG GCC CTC CTC TAC TGG TTC TTG GTC CAG TCG GAC TGG ACG GAC CAGThr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val361_____________365_____________________hFC DOMAIN______375_________________380            1150        1160            1170            1180            1190            1200             |             |               |              |               |           |AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AACTTT CCG AAG ATA GGG TCG CTG TAG CGG CAC CTC ACC CTC TCG TTA CCC GTC GGC CTC TTGLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn381 ____________385_____________________hFC DOMAIN______395_________________400            1210        1220            1230            1240            1250            1260             |             |               |              |               |           |AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AAGTTG ATG TTC TGG TGC GGA GGG CAC GAC CTG AGG CTG CCG AGG AAG AAG GAG ATG TCG TTCAsn Tyr Lys Tyr Tyr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys401_____________405_____________________hFC DOMAIN______415_________________420            1270        1280            1290            1300            1310            1320             |             |               |              |               |           |CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CATGAG TGG CAC CTG TTC TCG TCC ACC GTC GTC CCC TTG CAG AAG AGT ACG AGG CAC TAC GTALys Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His421_____________425_____________________hFC DOMAIN______435_________________440            1330        1340            1350           1360            1370            1377              |             |               |              |               |           |GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA TGA(SEQ ID NO: 5)CTC CGA GAC GTG TTG GTG ATG TGC GTC TTC TCG GAG AGG GAC AGA GGC CCA TTT ACTGlu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys ***(SEQ ID NO: 6)441_____________445_____________________hFC DOMAIN______455_____________459

TABLE 4 VEGF-Grab3            10             20             30              40             50            60             |              |              |               |              |           |ATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CTG TGC GCG CTG CTC AGC TGT CTG CTT CTCTAC CAG TCG ATG ACC CTG TGG CCC CAG GAC GAC ACG CGC GAC GAG TCG ACA GAC GAA GAGMet Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser Cys Leu Leu Leu1_______________5__________hVEGFR1 SIGNAL SEQUENCE______15__________________20            70             80             90              100            110            120             |              |              |               |              |           |ACA GGA TCT AGT TCA GGT GAA TTC GGT AGA CCT TTC GTA GAG ATG TAC AGT GAA ATC CCCTGT CCT AGA TCA AGT CCA CTT AAG CCA TCT GGA AAG CAT CTC TAC ATG TCA CTT TAG GGGThr Gly Ser Ser Ser Gly Glu Phe Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro21______________25__26  27__________VEGFR1 DOMAIN 2_______35________________40            130           140            150             160            170            180             |              |              |               |              |           |GAA ATT ATA CAC ATG ACT GAA GGA AGG GAG CTC GTC ATT CCC TGC CGG GTT ACG TCA CCTCTT TAA TAT GTG TAC TGA CTT CCT TCC CTC GAG CAG TAA GGG ACG GCC CAA TGC AGT GGAGlu Ile Ile His Met Tyr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro41______________45__________________VEGFR1 DOMAIN 2_____55__________________60            190           200            210             220            230            240             |              |              |               |              |           |AAC ATC ACT GTT ACT TTA AAA AAG TTT CCA CTT GAC ACT TTG ATC CCT GAT GGA AAA CGCTTG TAG TGA CAA TGA AAT TTT TTC AAA GGT GAA CTG TGA AAC TAG GGA CTA CCT TTT GCGAsn Ile Thr Val The Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg61______________65__________________VEGFR1 DOMAIN 2_____75__________________80            250           260            270             280            290            300             |              |              |               |              |           |ATA ATC TGG GAC AGT AGA AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA GAA ATA GGGTAT TAG ACC CTG TCA TCT TTC CCG AAG TAG TAT AGT TTA CGT TGC ATG TTT CTT TAT CCCIle Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly81______________85__________________VEGFR1 DOMAIN 2_________95______________100            310           320            330             340            350            360             |              |              |               |              |           |CTT CTG ACC TGT GAA GCA ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT CTC ACA CATGAA GAC TGG ACA CTT CGT TGT CAG TTA CCC GTA AAC ATA TTC TGT TTG ATA GAG TGT GTALeu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His101_________VEGFR1 DOMAIN 2_________110_111 112_______VEGFR1 DOMAIN 3_______120            370           380            390             400            410            420             |              |              |               |              |           |CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA AGC CCA GTC ACA TTA CTTGCT GTT TGG TTA TGT TAG TAT CTA CAG GTT TAT TCG TGT GGT TCG GGT CAG TGT AAT GAAArg Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Ser Pro Val Thr Leu Leu121_____________125_________________VEGFR1 DOMAIN 3_____135_________________140            430           440            450             460            470            480             |              |              |               |              |           |AGA GGC CAT ACT CTT GTC CTC AAT TGT ACT GCT ACC ACT CCC TTG AAC ACG AGA GTT CAATCT CCG GTA TGA GAA CAG GAG TTA ACA TGA CGA TGG TGA GGG AAC TTG TGC TCT CAA GTTArg Gly His Thr Leu Val Leu Asn Cys Thr Arg Thr Thr Pro Leu Asn Thr Arg Val Gln141_____________145_________________VEGFR1 DOMAIN 3_____155_________________160            490          500             510             520            530            540             |              |              |               |              |           |ATG ACC TGG AGT TAC CCT GAT GAA AAA AAT AAG AAC GCT TCC GTA AGG CGA CGA ATT GACTAC TGG ACC TCA ATG GGA CTA CTT TTT TTA TTC TTG CGA AGG CAT TCC GCT GCT TAA CTGMet Thr Trp Ser Tyr Pro Asp Glu Lya Asn Lys Asn Ala Ser Val Arg Arg Arg Ile Asp161_____________165_________________VEGFR1 DOMAIN 3_____175_________________180            550          560             570             580            590            600             |              |              |               |              |           |CAA AGC AAT TCC CAT GCC AAC ATA TTC TAC AGT GTT CTT ACT ATT GAC AAA ATG CAG AACGTT TCG TTA AGG GTA CGG TTG TAT AAG ATG TCA CAA GAA TGA TAA CTG TTT TAC GTC TTGGln Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn181_____________185_________________VEGFR1 DOMAIN 3_____195_________________200            610          620             630             640            650            660             |              |              |               |              |           |AAA GAC AAA GGA CTT TAT ACT TGT CGT GTA AGG AGT GGA CCA TCA TTC AAA TCT GTT AACTTT CTG TTT CCT GAA ATA TGA ACA GCA CAT TCC TCA CCT GGT AGT AAG TTT AGA CAA TTGLys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys Ser Val Asn201_____________205_________________VEGFR1 DOMAIN 3_____215_________________220            670          680             690             700            710            720             |              |              |               |              |           |ACC TCA GTG CAT ATA TAT GAT AAA GCA CTC GAG GAC AAA ACT CAC ACA TGC CCA CCG TGCTGG AGT CAC GTA TAT ATA CTA TTT CGT GAG CTC CTG TTT TGA GTG TGT ACG GGT GGC ACGThr Ser Val His Ile Tyr Asp Lys Ala Leu Glu Asp Lys Thr His Thr Cys Pro Pro Cys221_________VEGFR1 DOMAIN 3_____229 230_____________hFC DOMAIN______________240            730          740             750             760            770            780             |              |              |               |              |           |CCA GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GACGGT CGT GGA CTT GAG GAC CCC CCT GGC AGT CAG AAG GAG AAG GGG GGT TTT GGG TTC CTGPro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp241_____________245_____________________hFC DOMAIN______255_________________260            790          800             810             820            830            840             |              |              |               |              |           |ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAATGG GAG TAC TAG AGG GCC TGG GGA CTC CAG TGT ACG CAC CAC CAC CTG CAC TCG GTG CTTThr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu261_____________265_____________________hFC DOMAIN______275_________________280            850          860             870             880            890            900             |              |              |               |              |           |GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACACTG GGA CTC CAG TTC AAG TTG ACC ATG CAC CTG CCG CAC CTC CAC GTA TTA CGG TTC TGTAsp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr281_____________285_____________________hFC DOMAIN______295_________________300            910          920             930             940            950            960             |              |              |               |              |           |AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTGTTC GGC GCC CTC CTC GTC ATG TTG TCG TGC ATG GCA CAC CAG TCG CAG GAG TGG CAG GACLys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu301_____________305_________________hFC DOMAIN__________315_________________320                         970          980             990            1000           1010            1020             |              |              |               |              |           |CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCAGTG GTC CTG ACC GAC TTA CCG TTC CTC ATG TTC ACG TTC CAG AGG TTG TTT CGG GAG GGTHis Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro321_____________325_____________________hFC DOMAIN______335_________________340            1030          1040          1050            1060            1070            1080             |              |              |               |              |           |GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TACCGG GGG TAG CTC TTT TGG TAG AGG TTT CGG TTT CCC GTC GGG GCT CTT GGT GTC CAC ATGAla Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr341_____________345_____________________hFC DOMAIN______355_________________360            1090          1100          1110            1120           1130            1140             |              |              |               |              |           |ACC CTG CCC CCA TCC CGG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTCTGG GAC GGG GGT AGG GCC CTC CTC TAC TGG TTC TTG GTC CAG TCG GAC TGG ACG GAC CAGThr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val361_____________365_____________________hFC DOMAIN______375_________________380            1150          1160          1170            1180           1190            1200             |              |              |               |              |           |AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AACTTT CCG AAG ATA GGG TCG CTG TAG CGG CAC CTC ACC CTC TCG TTA CCC GTC GGC CTC TTGLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn381_____________385_____________________hFC DOMAIN______395_________________400            1210          1220          1230            1240           1250            1260             |              |              |               |              |           |AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AAGTTG ATG TTC TGG TGC GGA GGG CAC GAC CTG AGG CTG CCG AGG AAG AAG GAG ATG TCG TTCAsn Tyr Lys Tyr Tyr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys401_____________405_____________________hFC DOMAIN______415_________________420            1270          1280          1290            1300           1310            1320             |              |              |               |              |           |CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CATGAG TGG CAC CTG TTC TCG TCC ACC GTC GTC CCC TTG CAG AAG AGT ACG AGG CAC TAC GTALys Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His421_____________425_____________________hFC DOMAIN______435_________________440            1330          1340          1350            1360       1370            1377             |              |              |               |          |           |GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA TGA (SEQ ID NO: 7)CTC CGA GAC GTG TTG GTG ATG TGC GTC TTC TCG GAG AGG GAC AGA GGC CCA TTT ACTGlu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys *** (SEQ ID NO: 8)441_____________445_____________________hFC DOMAIN______455_____________459

TABLE 5 Primer sequences for site-directed mutagenesis Mutation sitesR135S/K138T Forward 5′-AGCACACCAAGCCCAGTCACAT For VEGF-Grab1 primerTACTTAGA-3′ (SEQ ID NO: 9) and VEGF-Grab3 Reverse5′-TCTAAGTAATGTGACTGGGCTT primer GGTGTGCT-3′ (SEQ ID NO: 10) R172NForward 5′-AATAAGAACGCTTCCGTAAGGC For VEGF-Grab2 primer GACGAATT-3′(SEQ ID NO: 11) and VEGF-Grab3 Reverse 5′-AATTCGTCGCCTTACGGAAGCG primerTTCTTATT-3′ (SEQ ID NO: 12)

TABLE 6 Primer sequences for Quantitative Real Time PCR GeneForward primer Reverse primer mGAPDH GTCGTGGAGTCTACTGGTGTCTTCACGTTGTCATATTTCTCGTGGTTCACACCC (SEQ ID NO: 13) (SEQ ID NO: 14) mVEGFAGTCAGAGAGCAACATCACCATGCAG CTTTGG TCTGCATTCACATCTGCTG (SEQ ID NO: 15)(SEQ ID NO: 16) mP1GF GATGCTGGTCATGAAGCTGTTC TCGTCTCC AGAATAGGTCTGCA(SEQ ID NO: 17) (SEQ ID NO: 18) mVEGF-C CGTTCTCTGCCAGCAACATTACCACCTTGTTGG GTCCACAGACATCATGG (SEQ ID NO: 19) (SEQ ID NO: 20) mPDGF-BCACAGAGACTCCGTAGATGAAGATGGG CA CTCGGCGATTACAGCAGGCTCTG (SEQ ID NO: 21)(SEQ ID NO: 22) mVEGFR1 GGCTCTACGACCTTAGACTGTCA TGCTGTTTCCTGGTCCTAAAATA(SEQ ID NO: 23) (SEQ ID NO: 24) mVEGFR2 ACCAGAAGTAAAAGTGATCCCAGATCCACCAAAAGATGGAGATAATTT (SEQ ID NO: 25) (SEQ ID NO: 26) mPDGFRαCGACTCCAGATGGGAGTTCCC TGCCATCCACTTCACAGGCA (SEQ ID NO: 27)(SEQ ID NO: 28) mPDGFRβ AGCTACATGGCCCCTTATGA GGAT CCCAAAAGACCAGACA(SEQ ID NO: 29) (SEQ ID NO: 30) mBv8 GCATGACAGGAGTCATCATTTTAAATGGCAGG ATATCAGGAAA (SEQ ID NO: 31) (SEQ ID NO: 32) mCCL2GAGCATCCACGTGTTGGCT TGGTGAATGAGTAGC AGCAGGT (SEQ ID NO: 33)(SEQ ID NO: 34)

TABLE 7 Antibodies Antibody Clone Source Primary Hamster anti-CD31 Clone2H8 Millipore FITC-conjugated mouse Clone 1A4 Sigma-Aldrich anti-α-SMARat anti-PDGFRβ APB5 eBioscience Rabbit anti-LYVE1 Polyclonal AngiobioRabbit anti-CD11b EP1345Y Millipore Rabbit anti-caspase3 Polyclonal R&DRabbit anti-collagen type IV Polyclonal Cosmobio Mouseanti-pan-cytokeratin AE1/AE3 Abcam Rat anti-cisplatin modified DNACP9/19 Abcam FITC-conjugated mouse 4.3.11.3 Hypoxyprobe, Incanti-Hypoxyprobe-1 Rabbit anti-phospho-VEGFR2 15D2 Cell Signaling Rabbitanti-VEGFR2 D5B1 Cell Signaling Rabbit anti-phospho-ERK1/2 PolyclonalCell Signaling Rabbit anti-ERK1/2 Polyclonal Cell Signaling SecondaryFITC-conjugated anti- Polyclonal Jackson ImmunoResearch hamster IgGCy3-conjugated anti- Polyclonal Jackson ImmunoResearch hamster IgGFITC-conjugated anti- Polyclonal Jackson ImmunoResearch rabbit IgGCy3-conjugated anti- Polyclonal Jackson ImmunoResearch rabbit IgGCy3-conjugated anti- Polyclonal Jackson ImmunoResearch rat IgGCy3-conjugated anti- Polyclonal Jackson ImmunoResearch mouse IgGCy3-conjugated anti- Polyclonal Jackson ImmunoResearch human Fc Goat Fabfragment anti- Polyclonal Jackson ImmunoResearch mouse IgGHRP-conjugated anti- Polyclonal Sigma Aldrich human Fc

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1. (canceled)
 2. The method according to claim 28, wherein the VEGFR1component comprises the second and third immunoglobulin (Ig)-likedomains.
 3. The method according to claim 2, wherein at least oneencoded positive amino acid residue in at least one domain of VEGFR1 ismutated to a negatively charged residue.
 4. The method according toclaim 3, wherein the domain is the third domain.
 5. The method accordingto claim 4, wherein the amino acid residue is on the β1-β2 loop, whichcomprises nucleic acid positions 397 to 432 of SEQ ID NO:1, whichcorresponds to amino acid residues 133 to 144 of SEQ ID NO:2, or β3-β4loop, which comprises nucleic acid positions 490 to 522 of SEQ ID NO:1,which corresponds to amino acid residues 164 to 174 of SEQ ID NO:2. 6.The method according to claim 28, wherein at least one encoded positiveamino acid residue in at least one domain of VEGFR1 is mutated so as toproduce a glycosylation site.
 7. The method according to claim 3,wherein at least one encoded positive amino acid residue in at least onedomain of VEGFR1 is mutated so as to produce decrease in net pI of theencoded polypeptide.
 8. The method according to claim 5, wherein theresidue to be mutated is R135 residue on the β1-β2 loop, K138 residue onthe β1-β2 loop, or R172 residue on the β3-β4 loop on the third domain.9. The method according to claim 1, wherein the VEGF is VEGF-A orVEGF-B.
 10. The method according to claim 28, wherein the VEGFR1component is operatively linked to a nucleotide sequence encoding amultimerizing component. 11.-27. (canceled)
 28. A method of blockingblood vessel growth in the eye such that the blood vessel growth causesa medical condition that affects sight in a mammal comprisingadministering to the mammal in need thereof an effective amount of apolypeptide encoded by an isolated nucleic acid molecule encoding apolypeptide capable of synchronously binding VEGF polypeptide andplacenta growth factor (PIGF) polypeptide comprising a nucleotidesequence encoding a VEGFR1 component. 29.-35. (canceled)
 36. The methodaccording to claim 28, wherein the medical condition is age-relatedmacular degeneration, exudative age-related macular degeneration,choroidal neovascularization, pathologic myopia, diabetic retinopathy,diabetic macular edema, retinal vein occlusion, retinopathy ofprematurity or neovascular glaucoma.
 37. The method according to claim36, wherein the choridal neovascularization is myopic choroidalneovascularization, traumatic choroidal neovascularization, uveiticchoroidal neovascularization, ocular histoplasmosis, or idiopathicchoroidal neovascularization. 38.-41. (canceled)